WO2021107155A1 - Procédé d'aide au diagnostic de la maladie de parkinson, biomarqueur, kit de réactifs et dispositif - Google Patents

Procédé d'aide au diagnostic de la maladie de parkinson, biomarqueur, kit de réactifs et dispositif Download PDF

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WO2021107155A1
WO2021107155A1 PCT/JP2020/044400 JP2020044400W WO2021107155A1 WO 2021107155 A1 WO2021107155 A1 WO 2021107155A1 JP 2020044400 W JP2020044400 W JP 2020044400W WO 2021107155 A1 WO2021107155 A1 WO 2021107155A1
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tetraspanin
amount
parkinson
disease
phosphatidylserine
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PCT/JP2020/044400
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English (en)
Japanese (ja)
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西部　隆宏
直子 今若
平安　一成
亮 請川
宏大 笹本
哲 小野寺
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富士フイルム和光純薬株式会社
富士フイルムワコーシバヤギ株式会社
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Priority to JP2021561586A priority Critical patent/JP7348604B2/ja
Priority to EP20892121.3A priority patent/EP4067905A4/fr
Priority to CN202080095136.9A priority patent/CN115190974A/zh
Publication of WO2021107155A1 publication Critical patent/WO2021107155A1/fr
Priority to US17/826,334 priority patent/US20220283146A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • the present invention relates to methods, biomarkers, reagent kits and devices that assist in the diagnosis of Parkinson's disease.
  • Parkinson's disease is a neurodegenerative disease in which nerve cells existing in the substantia nigra in the brain are degenerated and the striatal dopamine is deficient, resulting in the inability to move smoothly.
  • the prevalence of Parkinson's disease is as high as 100 to 150 per 100,000, and the prevalence increases with aging, so the number of patients is rapidly increasing with the aging of the population.
  • Parkinson's disease there is no cure for Parkinson's disease, but it is possible to improve motor symptoms by administering dopamine precursor therapeutic agents such as levodopa (L-Dopa) and dopamine agonists to supplement the reduced dopaminergic effect in the brain.
  • dopamine precursor therapeutic agents such as levodopa (L-Dopa) and dopamine agonists to supplement the reduced dopaminergic effect in the brain.
  • L-Dopa levodopa
  • dopamine agonists to supplement the reduced dopaminergic effect in the brain. Therefore, there is a need for a biomarker that can objectively determine the pathophysiology of Parkinson's disease.
  • Non-Patent Document 1 As a method for diagnosing Parkinson's disease, nerve-derived extracellular vesicles in blood are concentrated using an anti-NCAM antibody or an anti-L1CAM antibody, and Parkinson's disease is determined using ⁇ -synuclein or the like contained in the extracellular vesicles as an index.
  • Patent Document 2 Non-Patent Document 1, Non-Patent Document 2.
  • the method of diagnosing Parkinson's disease using nerve-derived extracellular vesicles in blood as an index requires an operation of affinity-concentrating nerve-derived extracellular vesicles from a sample, which is complicated.
  • an error is likely to occur between the assays, and it is difficult to apply it to the measurement of multiple samples.
  • the present invention relates to the following methods, biomarkers, reagent kits and devices for assisting the diagnosis of Parkinson's disease.
  • the determination of Parkinson's disease is based on the amount of extracellular vesicles having phosphatidylserine and tetraspanin, or the amount of extracellular vesicles having phosphatidylserine and tetraspanin relative to the amount of extracellular vesicles having tetraspanin.
  • the method for assisting the diagnosis of Parkinson's disease according to [1], wherein the subject is determined to have Parkinson's disease when the ratio is equal to or less than a reference value.
  • the measurement of the amount of the extracellular vesicle is to measure the amount of the extracellular vesicle having the phosphatidylserine and tetraspanin.
  • the determination of Parkinson's disease is to determine that the subject has Parkinson's disease by using the amount of extracellular vesicles having phosphatidylserine and tetraspanin as an index.
  • the method for assisting the diagnosis of Parkinson's disease according to [1] or [2], wherein the tetraspanin is selected from CD9, CD63, and CD81.
  • To measure the amount of the extracellular vesicle is to measure the amount of the extracellular vesicle having phosphatidylserine and tetraspanin. Measurement of the amount of extracellular vesicles having phosphatidylserine and tetraspanin is performed using a substance having an affinity for tetraspanin and a substance having an affinity for phosphatidylserine and extracellularly having phosphatidylserine and tetraspanin.
  • the determination of Parkinson's disease is to determine that the subject has Parkinson's disease using the amount of extracellular vesicles having phosphatidylserine and tetraspanin as an index, whichever is selected from [1] to [3].
  • the measurement of the amount of the extracellular vesicle is to measure the amount of the extracellular vesicle having the phosphatidylserine and tetraspanin.
  • the determination of Parkinson's disease is to determine that the subject has Parkinson's disease using the extracellular vesicles having phosphatidylserine and tetraspanin as an index.
  • the method for assisting the diagnosis of Parkinson's disease according to any one selected from [1] to [5], wherein the biological sample is a blood sample or cerebrospinal fluid.
  • the measurement of the amount of extracellular vesicles is to measure the amount of extracellular vesicles having phosphatidylserine and tetraspanin and the amount of extracellular vesicles having tetraspanin.
  • the determination of Parkinson's disease is to determine that the subject has Parkinson's disease by using the ratio of the amount of phosphatidylserine and the amount of extracellular vesicles having tetraspanin to the amount of extracellular vesicles having tetraspanin as an index.
  • the method of assisting the diagnosis of Parkinson's disease according to [1] or [2], wherein the tetraspanin is CD9 or CD63.
  • the measurement of the amount of extracellular vesicles is to measure the amount of extracellular vesicles having phosphatidylserine and tetraspanin and the amount of extracellular vesicles having tetraspanin. Measuring the amount of extracellular vesicles having phosphatidylserine and tetraspanin has said phosphatidylserine and tetraspanin using a substance having an affinity for tetraspanin and a substance having an affinity for phosphatidylserine.
  • the determination of Parkinson's disease is to determine that the subject has Parkinson's disease by using the ratio of the amount of phosphatidylserine and the amount of extracellular vesicles having tetraspanin to the amount of the extracellular vesicles having tetraspanin as an index.
  • the method for assisting the diagnosis of Parkinson's disease according to any one of 1], [2] and [7].
  • the method for assisting the diagnosis of Parkinson's disease according to [8], wherein the substance having an affinity for phosphatidylserine is a T cell immunoglobulin / mucin-containing protein.
  • the measurement of the amount of extracellular vesicles is to measure the amount of extracellular vesicles having phosphatidylserine and tetraspanin, and the amount of extracellular vesicles having tetraspanin.
  • the determination of Parkinson's disease is to determine that the subject has Parkinson's disease by using the ratio of the amount of phosphatidylserine and the amount of extracellular vesicles having tetraspanin to the amount of extracellular vesicles having tetraspanin as an index.
  • the method for assisting the diagnosis of Parkinson's disease according to any one selected from [1] to [2] and [7] to [9], wherein the biological sample is a blood sample or cerebrospinal fluid.
  • the determination of Parkinson's disease is based on the ratio of the amount of phosphatidylserine and the amount of extracellular vesicles having tetraspanin to the amount of extracellular vesicles having tetraspanin as an index, and the subject has Parkinson's disease and cognitive symptoms.
  • the diagnosis of Parkinson's disease according to any one selected from [1] to [2] and [7] to [10], which is to determine which of the Parkinson's diseases without the disease. How to assist.
  • a reagent kit for assisting diagnosis of Parkinson's disease which comprises a substance having an affinity for tetraspanin and a substance having an affinity for phosphatidylserine.
  • a biomarker for assisting the diagnosis of Parkinson's disease which comprises extracellular vesicles having phosphatidylserine and tetraspanin.
  • a device for assisting the diagnosis of Parkinson's disease which has a determination unit for determining that the disease is.
  • a measuring unit for measuring the amount of extracellular vesicles having phosphatidylserine and tetraspanin and the amount of extracellular vesicles having tetraspanin in a biological sample derived from a subject It has an arithmetic unit that calculates the ratio of the amount of phosphatidylserine and extracellular vesicles having tetraspanin to the amount of extracellular vesicles having tetraspanin, and phosphatidylserine and tetraspanin to the amount of extracellular vesicles having tetraspanin.
  • a device for assisting the diagnosis of Parkinson's disease which has a determination unit for determining that the subject has Parkinson's disease using the ratio of the amount of extracellular vesicles as an index.
  • a biomarker set for assisting the diagnosis of Parkinson's disease which comprises extracellular vesicles having phosphatidylserine and tetraspanin, and extracellular vesicles having tetraspanin.
  • the diagnosis of Parkinson's disease can be easily assisted.
  • FIG. 1 is a box whiskers graph in which plasma samples derived from Parkinson's disease patients without cognitive symptoms and plasma samples derived from healthy subjects were evaluated using the amount of exosomes having phosphatidylserine and CD9 as an index.
  • FIG. 2 is a box whiskers graph evaluating plasma samples derived from Parkinson's disease patients without cognitive symptoms and plasma samples derived from healthy subjects using the ratio of the amount of phosphatidylserine and the amount of exosomes having CD9 to the amount of extracellular vesicles having CD9 as an index. is there.
  • FIG. 1 is a box whiskers graph in which plasma samples derived from Parkinson's disease patients without cognitive symptoms and plasma samples derived from healthy subjects were evaluated using the amount of exosomes having phosphatidylserine and CD9 as an index. is there.
  • FIG. 2 is a box whiskers graph evaluating plasma samples derived from Parkinson's disease patients without cognitive symptoms and plasma samples derived from healthy subjects using the ratio of the amount of phosphatid
  • FIG. 3 shows a plasma sample derived from a Parkinson's disease patient without cognitive symptoms, a plasma sample derived from a Parkinson's disease patient with cognitive symptoms, and healthy using the ratio of the amount of phosphatidylserine and the amount of exosomes having CD9 to the amount of extracellular vesicles having CD9 as an index. It is a box whiskers graph which evaluated the plasma sample derived from a person.
  • FIG. 4 is a box-and-whisker graph in which plasma samples derived from Parkinson's disease patients and plasma samples derived from healthy subjects were evaluated using the amount of exosomes having phosphatidylserine and CD9 as an index.
  • 5 is a box whiskers graph in which plasma samples derived from Parkinson's disease patients and plasma samples derived from healthy subjects were evaluated using the ratio of the amounts of phosphatidylserine and exosomes having CD9 to the amount of extracellular vesicles having CD9 as an index.
  • Extracellular vesicles are small membrane vesicles derived from cells and composed of lipid bilayer membranes.
  • the extracellular vesicle include those having a diameter of 20 nm to 1000 nm, preferably those having a diameter of 50 nm to 800 nm, more preferably 50 nm to 500 nm, and particularly preferably 50 nm to 200 nm.
  • Examples of the extracellular vesicles include Nature Reviews Immunology 9, 581-593 (March 2009), "Obesity Research" Vol. 13 No. 2 2007 Topics As described in Naoto Aoki and others, those classified in various ways according to their origin and the size of small membrane vesicles can be mentioned.
  • exosomes include exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, apoptotic bodies, adiposomes and the like, with exosomes and vesicles being preferred, and exosomes being more preferred.
  • the exosome is a cell-derived small membrane vesicle composed of a lipid bilayer membrane, and examples thereof include those having a diameter of 50 nm to 200 nm, preferably 50 nm to 150 nm, and 50 nm to 100 nm. The one is more preferable. Exosomes are thought to be derived from late endosomes.
  • the microvesicles are cell-derived small membrane vesicles composed of a lipid bilayer membrane, and examples thereof include those having a diameter of 100 nm to 1000 nm, preferably 100 nm to 800 nm, and 100 nm to 500 nm. Is more preferable.
  • the microvesicles are thought to be derived from the cell membrane.
  • the extracellular vesicle may be contained in a biological sample derived from a subject or isolated from a biological sample derived from a subject, and may be isolated from a biological sample derived from a subject. It is preferable that the sample is made.
  • the biological sample derived from the subject may be any biological sample that can contain extracellular vesicles, for example, blood-derived samples such as serum, plasma, whole blood, and buffy coat, cerebrospinal fluid, urine, saliva, and semen. , Chest exudate, tears, sputum, mucus, lymph, ascites, pleural fluid, sheep water, bladder lavage fluid, bronchial alveolar lavage fluid, and other body fluid samples, including blood-derived samples and cerebrospinal fluid, preferably serum, plasma, and brain. Cerebrospinal fluid is more preferable, serum and plasma are more preferable, and plasma is particularly preferable. In addition, blood-derived samples are more useful because the burden of sampling on the subject is light.
  • blood-derived samples are more useful because the burden of sampling on the subject is light.
  • the biological sample derived from the subject for example, even if it is directly collected from the subject, has undergone pretreatment such as recovery, concentration, purification, isolation, dilution with a buffer solution, and filtration sterilization. You may. These pretreatments may be appropriately performed according to a conventional method.
  • the biological sample derived from the subject may be abbreviated as "biological sample”.
  • the method for isolating extracellular vesicles from the biological sample may be carried out according to a conventional method, and is not particularly limited.
  • Examples of the method for isolating extracellular vesicles from the biological sample include an affinity method (for example, PS affinity method), a fractional centrifugation method (for example, a pellet-down method, a chromatographic method, a density gradient centrifugation method, etc.).
  • Ultracentrifugation immunoprecipitation, chromatography (eg ion exchange chromatography, gel permeation chromatography), density gradient method (eg sucrose density gradient method), electrophoresis (eg organella electrophoresis) Method), magnetic separation method (for example, magnetically activated cell sorting (MACS) method), ultracentrifugation concentration method (for example, nanomembrane ultracentrifugation concentration method), percor gradient isolation method, method using a microfluidic device. , PEG precipitation method, etc., and the affinity method is preferable because extracellular vesicles having a high degree of purification can be obtained, or the fractional centrifugation method is preferable because theoretically unbiased recovery is possible.
  • chromatography eg ion exchange chromatography, gel permeation chromatography
  • density gradient method eg sucrose density gradient method
  • electrophoresis eg organella electrophoresis
  • magnetic separation method for example, magnetically activated cell sorting (
  • the ultracentrifugation method is more preferable, and the affinity method is particularly preferable.
  • the affinity methods the PS affinity method, which is an affinity purification for phosphatidylserine, is preferable.
  • the affinity method and the fractional centrifugation method may be performed according to, for example, the method described in WO2016 / 0886689. As these isolation methods, only one kind may be used, or two or more kinds may be combined. In addition, isolation by one isolation method may be repeated twice or more.
  • the subject is not particularly limited, and for example, a person diagnosed with Parkinson's disease based on the diagnostic criteria, a person diagnosed with the risk of developing Parkinson's disease based on the diagnostic criteria, and a person diagnosed with Parkinson's disease.
  • Those who have not, those who have not been diagnosed with Parkinson's disease based on diagnostic criteria, or those who have not been diagnosed as at risk of developing Parkinson's disease based on diagnostic criteria include those who have not been diagnosed with Parkinson's disease based on diagnostic criteria.
  • People who have been diagnosed with the disease, those who have been diagnosed at risk of developing Parkinson's disease based on diagnostic criteria, and those who have not been diagnosed with Parkinson's disease are preferred.
  • the diagnostic criteria include, for example, interviews, tests for Parkinson's disease biomarkers recommended by Parkinson's disease medical treatment guidelines such as MIBG myocardial scintigraphy and dopamine transporter scintigraphy, and tests for candidate substances for Parkinson's disease biomarkers. Based on the results of the above, there are diagnostic criteria and the like used when diagnosing Parkinson's disease.
  • the present invention relates to methods, biomarkers, reagent kits and devices that assist in the diagnosis of Parkinson's disease.
  • a biomarker containing extracellular vesicles having phosphatidylserine and tetraspanin is used, and the amount of extracellular vesicles having phosphatidylserine and tetraspanin is used as an index to determine that the subject has Parkinson's disease.
  • the first invention an extracellular vesicle having phosphatidylserine and tetraspanin, and a biomarker containing an extracellular vesicle having tetraspanin are used in combination, and the cell having the tetraspanin.
  • the subject is determined to have Parkinson's disease using the ratio of the amount of phosphatidylserine and tetraspanin-containing extracellular vesicles to the amount of external vesicles as an index (hereinafter, may be abbreviated as "second invention”). Is included.
  • the biomarker for assisting the diagnosis of Parkinson's disease in the first invention includes extracellular vesicles having phosphatidylserine and tetraspanin.
  • the extracellular vesicles in the PD marker are similar to those described above, as are the preferred ones.
  • the extracellular vesicles having phosphatidylserine and tetraspanin in the PD marker have at least one tetraspanin such as CD9, CD63, CD81, CD151 and phosphatidylserine which is a phospholipid on the membrane surface, and CD9, CD63, and CD81.
  • Those having at least one tetraspanin selected from CD9 and phosphatidylserine are preferable, those having at least one tetraspanin selected from CD9 and CD81 and phosphatidylserine are more preferable, and those having CD9 and phosphatidylserine are particularly preferable.
  • the method for assisting the diagnosis of Parkinson's disease in the first invention (hereinafter, may be abbreviated as "PD diagnosis assisting method”) is to measure the amount of extracellular vesicles having phosphatidylserine and tetraspanin in a biological sample. This includes determining that the subject has Parkinson's disease by using the amount of extracellular vesicles having phosphatidylserine and tetraspanin in the biological sample as an index.
  • the biological sample, subject, and extracellular vesicle in the PD diagnosis assisting method are the same as those described above, and the preferred ones are also the same.
  • the extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnostic assisting method are the same as those described above in the PD marker, and the preferred ones are also the same.
  • the “amount” in the PD diagnosis assisting method includes mass and concentration. Further, the “amount” includes actually measured values having a correlation with mass and concentration (for example, absorbance, amount of change in absorbance, transmitted light, amount of change in transmitted light, fluorescence intensity, amount of change in fluorescence intensity, amount of light emission, change in amount of light emission). Amount, turbidity, turbidity change rate, scattered light, scattered light change rate, reflectance, reflectance change amount, refractive index, refractive index change amount, etc.) are also included.
  • the measurement of the amount of extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnostic assist method is only the amount of extracellular vesicles having one type of tetraspanin and phosphatidylserine (eg, extracellular vesicles having CD9 and phosphatidylserine).
  • the amount of extracellular vesicles having two or more tetraspanins and phosphatidylserine (eg, extracellular vesicles having CD9 and phosphatidylserine, and extracellular vesicles having CD81 and phosphatidylserine) May be measured, and it is preferable to measure only the amount of extracellular vesicles having one type of tetraspanin and phosphatidylserine.
  • the measurement of the amount of extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnostic assisting method is not particularly limited as long as it is a method usually performed in this field.
  • an immunological measurement method using a substance having an affinity for tetraspanin and a substance having an affinity for phosphatidylserine, a mass analysis method, a method combining these methods, or the like may be used.
  • an immunological measurement method using a substance having an affinity for tetraspanin and a substance having an affinity for phosphatidylserine is preferable.
  • the immunological measurement method includes a method using an immune reaction (antigen-antibody reaction) and a method using a binding force between two molecules other than the antigen-antibody reaction such as binding of a lectin and a protein (immunity).
  • a method based on the scientific measurement method) is also included.
  • the substance having an affinity for tetraspanin may be a substance that specifically binds to tetraspanin, for example, an antibody that specifically binds to tetraspanin or a sugar chain possessed by tetraspanin.
  • examples thereof include proteins that specifically bind to tetraspanin such as lectin that specifically binds to tetraspanin, and nucleic acids that specifically bind to tetraspanin. Proteins that specifically bind to tetraspanin are preferable, and proteins that specifically bind to tetraspanin are preferable. Antibodies that specifically bind are more preferred.
  • Examples of the antibody that specifically binds to tetraspanin include anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody, anti-CD151 antibody, and anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody are preferable.
  • CD9 antibody and anti-CD81 antibody are more preferable, and anti-CD9 antibody is particularly preferable.
  • As the substance having an affinity for tetraspanin only one kind may be used, or two or more kinds may be used, and it is preferable to use only one kind.
  • the substance having an affinity for phosphatidylserine may be a substance that specifically binds to phosphatidylserine.
  • a protein that specifically binds to phosphatidylserine examples thereof include nucleic acids that specifically bind to phosphatidylserine, and proteins that specifically bind to phosphatidylserine are preferable.
  • the protein that specifically binds to phosphatidylserine include an antibody that specifically binds to phosphatidylserine and a phosphatidylserine-affinity protein, and a phosphatidylserine-affinity protein is preferable.
  • Examples of the antibody that specifically binds to phosphatidylserine include anti-phosphatidylserine antibody 1H6 (Merck Co., Ltd.).
  • Examples of the phosphatidylserine-affinitive protein include Tim1 (T-cell immunoglobulin / mutin domain-containing molecule 1, T-cell antibody / mucin-domain 1), Tim2 (T-cell immunoglobulin / mutin domain-containing molecule 2, T-cell).
  • Tim3 T-cell immunoglobulin / mutin domain-containing molecule 3, T-cell antibody-mucin-domain 3
  • Tim4 T-cell immunoglobulin / mutin domain-containing molecule 4, T-cell immunoglobulin
  • Tim protein anti-phosphatidylserine antibody
  • Annexin V Annexin V
  • Tim protein, anti-phosphatidylserine antibody are preferable, and Tim protein is more preferable.
  • Tim protein is more preferable.
  • Tim protein As the Tim protein, Tim1 and Tim4 are preferable, and Tim4 is more preferable.
  • the substance having an affinity for the phosphatidylserine only one kind may be used, or two or more kinds may be used, and it is preferable to use only one kind.
  • the substance having an affinity for the tetraspanin and the substance having an affinity for the phosphatidylserine may be a commercially available product or a substance appropriately prepared by a conventional method.
  • the antibody that specifically binds to the tetraspanin and the antibody that specifically binds to the phosphatidylserine may be either a polyclonal antibody or a monoclonal antibody, and these may be used alone or in combination thereof. May be used.
  • the antibody that specifically binds to the tetraspanin and the antibody that specifically binds to the phosphatidylserine are not only the immunoglobulin molecule itself (intact immunoglobulin) but also a fragment thereof and the ability to bind to an antigen.
  • fragment antibodies such as Fab, F (ab') 2, F (ab'), single chain antibodies (single chain Fv), and synthetic antibodies such as diabody, tribody, and tetrabody, which are fragments having May be good.
  • these antibodies may be prepared according to the method described in, for example, "Immunoassay" (edited by Biochemical Measurement Study Group, Kodansha, 2014).
  • the substance having an affinity for tetraspanin and / and the substance having an affinity for phosphatidylserine may be labeled with a labeling substance.
  • the labeling substance include enzymes such as peroxidase, microperoxidase, and alkaline phosphatase, radioactive isotopes such as 99mTc, 131I, 125I, 14C, 3H, 32P, and 35S, fluorescein, fluorescein isothiocyanate (FITC), 4-.
  • Fluorescent substances such as methylumveriferone, HiLyte, Alexa, CyDye or rhodamine, or derivatives thereof, luminescent substances such as luciferin, luminol, ruthenium complex, phenol, naphthol, or anthracene, or ultraviolet rays such as derivatives thereof.
  • Substances that have absorption into fluorescein substances that have properties as spin labeling agents typified by compounds having an oxyl group such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, gold colloids, Examples thereof include nanoparticles such as quantum dots, and enzymes and fluorescent substances are preferable.
  • the method for labeling the substance having an affinity for the tetraspanin and / or the substance having an affinity for the phosphatidylserine with the labeling substance is not particularly limited, and may be carried out according to a labeling method known per se.
  • the measurement of these labeling substances may be carried out according to a measurement method known per se according to the labeling substance.
  • the substance having an affinity for the tetraspanin and the substance having an affinity for the phosphatidylserine have either of these or one of them as the primary affinity substance, and specifically bind to the primary affinity substance 2
  • Secondary affinity substances eg, secondary antibodies
  • the secondary affinity substance may be labeled with a labeling substance, preferably labeled, and more preferably a secondary antibody labeled with the labeling substance.
  • labeling substance and the labeling method are the same as those described above, and the preferred ones are also the same.
  • labeling with a labeling substance is a substance having an affinity for the tetraspanin and / or a substance having an affinity for the phosphatidylserine to which one of avidins and biotins is bound, and a labeling substance.
  • the binding of avidins and biotins may be utilized by using the binding of the remaining one of avidins and biotins.
  • biotins include biotin, iminobiotin, desthiobiotin, biocitin, biotin sulfokide and the like, and biotin is preferable.
  • avidins include avidin, tamavidin, tamavidin 2, streptavidin and the like, and streptavidin is preferable.
  • the method of combining may be carried out according to a conventional method.
  • the substance having an affinity for tetraspanin and / and the substance having an affinity for the phosphatidylserine may be immobilized on the solid phase, and the substance having an affinity for phosphatidylserine may be immobilized on the solid phase. It is preferably fixed.
  • solid phase examples include synthetic polymer compounds such as latex, polystyrene, polypropylene, polyacrylic acid, polymethacrylic acid, polyacrylamide, polyglycidyl methacrylate, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, and silicone rubber.
  • synthetic polymer compounds such as latex, polystyrene, polypropylene, polyacrylic acid, polymethacrylic acid, polyacrylamide, polyglycidyl methacrylate, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, and silicone rubber.
  • inorganic substances such as porous glass, styrene glass, ceramics, alumina, silica gel, activated carbon and metal oxides. Further, two or more of these may be used in combination.
  • the shape of the solid phase is not particularly limited, and for example, a microtiter plate (ELISA plate), beads, tubes (microtubes), particles, a dedicated tray in which a large number of tubes are integrally molded, a disk-shaped piece, a test tube, etc. Can be mentioned.
  • ELISA plate microtiter plate
  • beads beads
  • tubes microtubes
  • particles a dedicated tray in which a large number of tubes are integrally molded
  • a disk-shaped piece a test tube, etc.
  • the method for immobilizing a substance having an affinity for tetraspanin and / or a substance having an affinity for phosphatidylserine on the solid phase is not particularly limited as long as it is a method usually used in this field. You can do it according to the usual method.
  • the combination of the substance having an affinity for the tetraspanin and the substance having an affinity for the phosphatidylserine is not particularly limited, but the protein that specifically binds to the tetraspanin and the phosphatidyl are not particularly limited.
  • a combination of proteins that specifically bind to serine is preferable, and a combination of an antibody that specifically binds to the tetraspanin and an antibody that specifically binds to the phosphatidylserine or the phosphatidylserine-affinity protein is more preferable. preferable.
  • an antibody that specifically binds to the tetraspanin and an antibody that specifically binds to phosphatidylserine or a phosphatidylserine-affinitive protein for example, an antibody that specifically binds to tetraspanin and a Tim protein or Examples thereof include a combination of antibodies that specifically bind to phosphatidylserine, preferably an anti-CD9 antibody, an anti-CD63 antibody, or a combination of an anti-CD81 antibody and a Tim protein, and a combination of an anti-CD9 antibody or an anti-CD81 antibody and a Tim protein.
  • the combination of anti-CD9 antibody or anti-CD81 antibody and Tim1 or Tim4 is more preferable, the combination of anti-CD9 antibody or anti-CD81 antibody and Tim4 is particularly preferable, and the combination of anti-CD9 antibody and Tim4 is most preferable.
  • ELISA method enzyme-bound immunoassay measurement method
  • EIA method enzyme immunoassay method
  • RIA radiation immunoassay method
  • Immunoassay method such as electrophoresis, western blot method, latex immunoassay (NIA method), immunoturbidimetric method such as latex immunoassay (TIA method), fine particle counting immunoaggregation measurement method (PCIA method) Immunoaggregation method such as, surface plasmon resonance method (SPR method), AlphaLISA method, assay for detecting the presence of target molecule using fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET), etc.
  • Immunoassays and mass analyzes include enzyme-bound immunoassays (ELISA), chemoluminescent enzyme immunoassays (CLEIA), chemoluminescence immunoassays (CLIA), electrochemical luminescence immunoassays.
  • the method (ECLIA method) and capillary electrophoresis are preferable, and the enzyme-bound immunoassay measurement method (ELISA method), chemoluminescent enzyme immunoassay (CLEIA method), and LBA-EATA method are more preferable, and the enzyme-bound immunoassay measurement method (ELISA method).
  • ELISA method is particularly preferable.
  • the principle for measuring the amount of extracellular vesicles having phosphatidylserine and tetraspanin is not particularly limited, and examples thereof include a sandwich method and a competitive method, and the sandwich method is preferable. Further, as the measurement principle, a homogenius method, a heterogeneous method or the like may be used, and the heterogeneous method is preferable.
  • the immunological measurement method for example, it has an affinity for the tetraspanin other than the antibody that specifically binds to the tetraspanin and the antibody that specifically binds to the phosphatidylserine.
  • examples thereof include a method of performing the immunological measurement method using a substance and a substance having an affinity for the phosphatidylserine, and the same method is also preferable.
  • the amount of extracellular vesicles having phosphatidylserine and tetraspanin may be specifically measured according to the method described in WO2016 / 0886689, and all the descriptions in the publication are incorporated in the present specification.
  • the measurement of the amount of extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnostic assist method specifically, for example, (1) for extracellular vesicles having phosphatidylserine and tetraspanin in a biological sample and tetraspanin.
  • an extracellular vesicle having phosphatidylserine and tetraspanin in a biological sample a substance having an affinity for tetraspanin, and a substance having an affinity for tetraspanin.
  • a step of forming a complex containing a substance having an affinity for phosphatidylserine (hereinafter, may be abbreviated as “complex formation step”), and (2) a step of measuring the amount of the complex (2).
  • a method including (may be abbreviated as “complex amount measuring step”) is mentioned as a preferable method.
  • the order in which the substance having an affinity for tetraspanin and the substance having an affinity for phosphatidylserine in the complex formation step and the biological sample are brought into contact with each other is not particularly limited, but the affinity for the biological sample and the phosphatidylserine It is preferable to contact the substance having an affinity for tetraspanin after contacting the substance having the substance. That is, the complex formation step is composed of an extracellular vesicle in the biological sample and a substance having an affinity for phosphatidylserine by contacting the biological sample with a substance having an affinity for phosphatidylserine.
  • the first step of forming the first complex and the contact of the first complex with a substance having an affinity for tetraspanin have an affinity for the first complex and tetraspanin. It is preferable to include a second step of forming a second complex composed of the material.
  • an antibody or Tim protein (preferably Tim1 or Tim4, more preferably Tim4) that specifically binds to phosphatidylserine immobilized on a solid phase and a biological sample are used.
  • an antibody or Tim protein that specifically binds to phosphatidylserine is formed into a first complex of phosphatidylserine and tetraspanin-bearing extracellular vesicles in a biological sample, and the first complex is formed.
  • an antibody that specifically binds to tetraspanin are brought into contact with each other to form a second complex of the first complex and an antibody that specifically binds to tetraspanin.
  • washing operation (B / F separation) at least before the complex amount measuring step.
  • the washing operation may be performed after forming the first complex and / or after forming the second complex in the above method, and after forming the first complex. It is preferable to perform the cleaning operation (B / F separation), and further perform the cleaning operation (B / F separation) after forming the second complex.
  • extracellular vesicles having phosphatidylserine and tetraspanin, a substance having an affinity for tetraspanin, and a substance having an affinity for phosphatidylserine obtained in the complex forming step were used. Any method may be used as long as it is a step of measuring the amount of the complex contained and the amount of the complex can be measured.
  • an antibody that specifically binds to tetraspanin labeled with a labeling substance is used, or (2) "specifically binds to tetraspanin.”
  • a labeled secondary antibody labeled with a labeling substance that specifically binds to the "antibody to be used” or (3) an antibody that specifically binds to tetraspanin to which one of avidins and biotins is bound. It specifically binds to the phosphatidylserine immobilized on the solid phase obtained in the above-mentioned complex formation step by using a labeling substance in which the other one of avidins and biotins is bound.
  • Antibodies and labeling substances that specifically bind to tetraspanin and extracellular vesicles having phosphatidylserine and tetraspanin in biological samples with antibodies or Tim proteins (preferably Tim1 or Tim4, more preferably Tim4).
  • the labeling substance in the complex containing preferably a sex substance may be detected.
  • the complex amount measurement step it is preferable to perform a washing operation (B / F separation) before detecting the labeling substance.
  • Measurement of the amount of extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnostic aid method is more specifically, for example, an antibody or Tim protein that specifically binds to phosphatidylserine immobilized on a solid phase plate. (Preferably Tim1 or Tim4, more preferably Tim4) and the biological sample are brought into contact with an antibody or Tim protein that specifically binds to phosphatidylserine and extracellular vesicles having phosphatidylserine and tetraspanin in the biological sample.
  • the first complex After forming a first complex with, and if necessary, B / F separation, (1) the first complex is brought into contact with an antibody that specifically binds to tetraspanin labeled with a labeling substance. , Forming a second complex of the first complex with an antibody that specifically binds to tetraspanin labeled with a labeling substance, (2) specific to the first complex and tetraspanin. The first complex is brought into contact with the antibody that specifically binds to form a second complex of the antibody that specifically binds to tetraspanin, and if necessary, after B / F separation, the second complex is formed.
  • the labeled secondary antibody labeled with a labeling substance that specifically binds to the "antibody that specifically binds to the tetraspanin” are brought into contact with the second complex and the labeled.
  • a third complex with the secondary antibody is formed, or (3) the first complex is brought into contact with an antibody that specifically binds to tetraspanin to which one of avidins and biotins is bound.
  • a second complex is formed between the first complex and an antibody that specifically binds to tetraspanin to which one of avidins and biotins is bound, and if necessary, after B / F separation, the said A third complex of a labeling substance (preferably an enzyme or a fluorescent substance) is formed by binding the second complex with the remaining one of avidins and biotins, and is obtained after B / F separation.
  • the labeling substance of the second complex or the third complex may be detected.
  • the amount (concentration) of the substance and the substance having an affinity for phosphatidylserine, the labeling substance, the labeling method, etc. are appropriately set according to the type of biological sample, the required measurement sensitivity, the measurement method to be used, the measurement device, and the like. do it.
  • the amount (concentration) of extracellular vesicles having phosphatidylserine and tetraspanin in a biological sample may be calculated by preparing a calibration curve using a standard product.
  • the standard product include extracellular vesicles having phosphatidylserine and tetraspanin.
  • the determination of Parkinson's disease in the PD diagnostic assist method includes determining Parkinson's disease using the amount of extracellular vesicles having phosphatidylserine and tetraspanin as an index.
  • the determination of Parkinson's disease includes, for example, determination of whether or not the subject is likely to have Parkinson's disease, determination of whether or not the subject is likely to have Parkinson's disease, and determination of whether or not the subject is likely to have Parkinson's disease.
  • Determining whether the subject is at high risk of developing Parkinson's disease determining whether the subject is at risk of developing Parkinson's disease, determining whether the subject is likely to have Parkinson's disease, and determining whether the subject is suffering from Parkinson's disease It is preferable to determine the presence or absence of the possibility of suffering from Parkinson's disease.
  • the determination of Parkinson's disease in the PD diagnostic assist method is determined, for example, by measuring the amount of extracellular vesicles having phosphatidylserine and tetraspanin described above, and extracellularly having phosphatidylserine and tetraspanin in a biological sample derived from a subject. It is done by comparing the amount of vesicles with a predetermined reference value (cutoff value). Specifically, if the amount of extracellular vesicles carrying phosphatidylserine and tetraspanin is below a predetermined reference value, it is highly likely that the subject has Parkinson's disease, or the subject has Parkinson's disease.
  • a predetermined reference value cutoff value
  • the subject is at high risk of developing Parkinson's disease, or that the subject is at high risk of developing Parkinson's disease.
  • the amount of extracellular vesicles having phosphatidylserine and tetraspanin is larger than a predetermined standard value, it is unlikely that the subject has Parkinson's disease or the subject has Parkinson's disease. It can be determined that there is no possibility that the subject has Parkinson's disease, the subject has a low risk of developing Parkinson's disease, or the subject has no risk of developing Parkinson's disease.
  • the predetermined reference value is the amount of extracellular vesicles having phosphatidylserine and tetraspanin in a biological sample, which is preset to distinguish between a Parkinson's disease patient and a healthy person.
  • the method for determining the predetermined reference value is not particularly limited, and for example, the amount of extracellular vesicles having phosphatidylserine and tetraspanin contained in a biological sample obtained from a patient suffering from Parkinson's disease and a healthy person is determined.
  • the amount of extracellular vesicles having phosphatidylserine and tetraspanin obtained by measurement it can be determined by statistical analysis such as ROC analysis (Receiver Operating Characteristic analysis).
  • ROC analysis Receiveiver Operating Characteristic analysis
  • the predetermined reference value can be set so that the sensitivity is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, and 90% or more. More preferably, for example, the specificity can be determined to be 60% or more, 70% or more is preferable, 80% or more is more preferable, and 90% or more is further preferable.
  • the reagent kit for assisting diagnosis of Parkinson's disease in the first invention (hereinafter, may be abbreviated as "reagent kit for assisting PD diagnosis”) has an affinity for a substance having an affinity for tetraspanin and an affinity for phosphatidylserine. Includes substances with.
  • the substances having an affinity for tetraspanin and the substances having an affinity for phosphatidylserine in the reagent kit for assisting PD diagnosis are the same as those described above in the method for assisting PD diagnosis, respectively, and the preferable substances are also the same. ..
  • the substance having an affinity for the tetraspanin and the substance having an affinity for the phosphatidylserine may be in a solution state, a frozen state, a dried state, or a freeze-dried state, respectively. Further, the substance having an affinity for the tetraspanin and the substance having an affinity for the phosphatidylserine may be included in the kit as one reagent or included in the kit as separate reagents. ..
  • the combination of the substance having an affinity for tetraspanin and the substance having an affinity for phosphatidylserine in the reagent kit for assisting PD diagnosis includes the same combinations as those described above in the method for assisting PD diagnosis, and preferred combinations are also available. The same is true.
  • the substance having an affinity for tetraspanin and / and the substance having an affinity for phosphatidylserine in the PD diagnostic aid reagent kit may be immobilized on a solid phase and may be labeled with a labeling substance. May be good.
  • the solid phase, the method for immobilizing the solid phase, the labeling substance, and the labeling method are the same as those described above in the PD diagnosis assisting method, and the preferred ones are also the same.
  • the PD diagnostic aid reagent kit is a secondary affinity substance (for example, secondary affinity substance) that specifically binds to a substance having an affinity for the tetraspanin and / and a substance having an affinity for the phosphatidylserine. Antibodies) may be further included.
  • the secondary affinity substance in the PD diagnosis assisting reagent kit is the same as that described above in the PD diagnosis assisting method, and the preferred one is also the same. Further, the secondary affinity substance in the PD diagnosis assisting reagent kit may be labeled with a labeling substance, and the labeling substance and the labeling method are the same as those described above in the PD diagnosis assisting method, which is preferable. The same is true for things.
  • the substance having an affinity for tetraspanin and / and the substance having an affinity for phosphatidylserine in the PD diagnostic aid reagent kit may be a substance to which one of avidins and biotins is bound, and the avidins. And the biotins are the same as those described above in the PD diagnosis assisting method, and the preferable ones are also the same.
  • the PD diagnostic assisting reagent kit may further contain a labeling substance to which one of avidins and biotins is bound, and the labeling substance and labeling method are the same as those described above in the PD diagnostic assisting method. The same applies to preferred ones.
  • the concentration (amount) of the substance having an affinity for tetraspanin and the substance having an affinity for phosphatidylserine in the PD diagnostic aid reagent kit is within the range usually used in this field depending on the measurement method. It may be set as appropriate.
  • the substance having an affinity for tetraspanin preferably contains, for example, a concentration at the time of use, which is usually 10 to 20000 ng / mL or 100 to 10000 ng / mL when immobilized on a solid phase. When used for detection, those containing an amount of 10 to 5000 ng / mL and 100 to 500 ng / mL are usually preferable.
  • the substance having an affinity for phosphatidylserine contains, for example, a concentration at the time of use, which is usually 10 to 20000 ng / mL or 100 to 10000 ng / mL when immobilized on a solid phase. Is preferable, and when it is used for detection, it is usually preferably one containing an amount of 10 to 5000 ng / mL and 100 to 500 ng / mL.
  • reagents usually used in this field such as buffers, reaction accelerators, sugars, proteins, salts, stabilizers such as surfactants, preservatives, etc., are used as substances having an affinity for tetraspanin. / And may coexist with a substance having an affinity for phosphatidylserine. These concentrations and pHs may be appropriately selected from the ranges usually used in this field.
  • the PD diagnostic aid reagent kit includes substances having an affinity for tetraspanin and substances having an affinity for phosphatidylserine, as well as extracellular tetraspanin and phosphatidylserine using these affinity substances. It may be equipped with the reagents necessary to measure the amount of vesicles. Examples of such reagents include cleaning agents, sample diluents (reagents for diluting samples), reagents for detecting labeling substances, reagents for binding labeling substances to these affinity substances, and the like. Examples thereof include reagents for immobilizing the affinity substances in the solid phase, and reagents for binding avidins or biotins to these affinity substances and labeling substances. The concentration, pH, etc. of these reagents may be appropriately selected from the range usually used in this field.
  • the PD diagnostic aid reagent kit may include a standard product used to prepare a calibration curve for extracellular vesicles having phosphatidylserine and tetraspanin.
  • Examples of the standard product include extracellular vesicles having phosphatidylserine and tetraspanin.
  • the standard product may be in a solution state, a frozen state, a dried state, or a freeze-dried state.
  • reagents usually used in this field such as buffers, reaction accelerators, sugars, proteins, salts, stabilizers such as surfactants, preservatives, etc., may coexist with the standard product. These concentrations and pHs may be appropriately selected from the ranges usually used in this field.
  • the PD diagnostic aid reagent kit may include package inserts and instruction manuals.
  • the package insert and instruction manual include, for example, measuring the amount of extracellular vesicles having phosphatidylserine and tetraspanin in a biological sample derived from a subject and / and extracellular vesicles having phosphatidylserine and tetraspanin. Examples include package inserts and instruction manuals that describe that the subject is determined to have Parkinson's disease using the amount as an index. These package inserts and instruction manuals may be described separately in a plurality of cases, or may be described collectively in one case.
  • the PD diagnostic aid reagent kit includes, for example, a substance in which either a substance having an affinity for tetraspanin or a substance having an affinity for phosphatidylserine is immobilized on a solid phase and tetraspanin.
  • the remaining one of the substance having an affinity for phosphatidylserine and the substance having an affinity for phosphatidylserine is bound to the labeling substance or the other affinity substance and the substance indirectly labeling this Examples include kits containing ingredients for binding to.
  • the PD diagnostic aid reagent kit includes a substance having an affinity for tetraspanin and a substance having an affinity for phosphatidylserine in which either one is immobilized on a solid phase and the following (1) to (3).
  • a kit containing any one of the above is preferred. (1) A substance having an affinity for tetraspanin and a substance having an affinity for phosphatidylserine in which the remaining one is bound to a labeling substance, (2) a substance having an affinity for tetraspanin and a substance having an affinity for phosphatidylserine.
  • Preferred specific examples of the reagent kit for assisting PD diagnosis include a solid phase plate on which an antibody or Tim protein that specifically binds to phosphatidylserine (Tim4 or Tim1 is more preferable, Tim4 is particularly preferable) and the following.
  • Examples thereof include a kit containing any one selected from (1) to (3).
  • a solution containing the other one of an antibody or Tim protein that specifically binds to phosphatidylserine usually 10-5000 ng / mL, 100). ⁇ 500 ng / mL is preferable
  • a solution usually 10 to 5000 ng / mL, 100 to 500 ng / mL
  • a secondary affinity substance that specifically binds to the affinity substance bound to the labeling substance.
  • a solution containing an antibody preferably anti-CD9 antibody or anti-CD81 antibody, more preferably anti-CD9 antibody
  • an antibody preferably anti-CD9 antibody or anti-CD81 antibody, more preferably anti-CD9 antibody
  • a solution containing a labeling substance to which the other one of avidins and biotins is bound usually 10-5000 ng / mL, preferably 100-500 ng / mL).
  • the device for assisting diagnosis of Parkinson's disease in the first invention (hereinafter, may be abbreviated as "device for assisting diagnosis of PD”) is the amount of extracellular vesicles having phosphatidylserine and tetraspanin in a biological sample derived from a subject. It has a measuring unit for measuring phosphatidylserine and tetraspanin, and a determining unit for determining that the subject has Parkinson's disease using the amount of extracellular vesicles having phosphatidylserine and tetraspanin as an index.
  • the biological sample, subject, and extracellular vesicle in the PD diagnosis assisting device are the same as those described above, and the preferred ones are also the same.
  • the measuring unit, the determination unit, and the like constituting the PD diagnosis assisting device may be arranged in the same device or may be separate from each other.
  • the measuring unit in the PD diagnostic assisting device is a site for measuring the amount of extracellular vesicles having phosphatidylserine and tetraspanin in the biological sample introduced into the device.
  • the size and configuration of the measuring unit are not particularly limited.
  • the measuring unit is, for example, an imaging device used for imaging using a microplate reader or CCD used in an ELISA method, an imaging device used in a method using a Western blotting method or a microarray (microchip), and a mass spectrometry method. Examples thereof include mass spectrometers, intermolecular interaction analyzers, flow cytometers used for flow cytometry, ultraviolet visible light detectors and fluorescence detectors used for HPLC methods and capillary electrophoresis methods.
  • the measurement target of the method for measuring the amount of extracellular vesicles having phosphatidylserine and tetraspanin and the amount of extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnostic assisting device is the same as that described above in the PD diagnostic assisting method. Yes, and the preferred ones are the same.
  • the measurement of extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnosis assisting device and the substances used for the measurement are the same as those described above in the PD diagnosis assisting method, and the preferred ones and specific examples are also the same.
  • the PD diagnosis assisting device may include a calculation unit.
  • the calculation unit in the PD diagnostic assistance device is an actually measured value (for example, absorbance, change in absorbance) that correlates with the mass and concentration of extracellular vesicles having phosphatidylserine and tetraspanin in the biological sample obtained by the measurement unit. , Transmitted light, transmitted light change amount, fluorescence intensity, fluorescence intensity change amount, light emission amount, light emission amount change amount, turbidity, turbidity change rate, scattered light, scattered light change rate, absorbance, reflectance change amount, refractive index It is a part that converts (rate, amount of change in refractive index, etc.) into mass, concentration, etc.
  • the actually measured value, the mass and the concentration converted by the calculation unit, and the like may be stored in a storage device or the like provided in the device such as a memory or a hard disk.
  • the determination unit in the PD diagnosis assisting device is a site for determining Parkinson's disease using the amount of extracellular vesicles having phosphatidylserine and tetraspanin obtained by the measurement unit or the calculation unit as an index.
  • the determination method of Parkinson's disease in the PD diagnosis assisting device, the predetermined reference value used for the determination, and the determination method of the predetermined reference value are the same as those described above in the PD diagnosis assisting method, and are preferable. The same applies to specific examples.
  • the predetermined reference value in the PD diagnosis assisting device may be stored in advance in the PD diagnosis assisting device, or may be input from the input site of the PD diagnosis assisting device at the time of determination.
  • the PD diagnosis assisting device may include an output unit.
  • the output unit performs processing such as displaying or outputting the determination result on a display device such as a display or a printing device such as a printer.
  • the biomarker set for assisting the diagnosis of Parkinson's disease in the second invention includes extracellular vesicles having phosphatidylserine and tetraspanin, and extracellular vesicles having tetraspanin. It is a combination of biomarkers.
  • the PD marker set for example, the ratio of the amount of phosphatidylserine and tetraspanin to the amount of extracellular vesicles having tetraspanin (hereinafter, may be abbreviated as "PD marker (II)").
  • PD marker (II) can be used as an index for determining that the subject has Parkinson's disease.
  • the PD marker (II) it can be used as an index for distinguishing whether the subject has Parkinson's disease with cognitive symptoms or Parkinson's disease without cognitive symptoms.
  • the extracellular vesicles in the PD marker set are the same as the extracellular vesicles, and the preferred ones are also the same.
  • the extracellular vesicles having phosphatidylserine and tetraspanin in the PD marker set have at least one tetraspanin such as CD9, CD63, CD81, CD151 and phosphatidylserine which is a phospholipid on the membrane surface, and CD9, CD63, and Those having at least one tetraspanin selected from CD81 and phosphatidylserine are preferable, those having at least one tetraspanin selected from CD9 and CD63 and phosphatidylserine are more preferable, and those having CD9 and phosphatidylserine are particularly preferable.
  • tetraspanin such as CD9, CD63, CD81, CD151 and phosphatidylserine which is a phospholipid on the membrane surface
  • CD9, CD63 Those having at least one tetraspanin selected from CD81 and phosphatidylserine are preferable, those having
  • Extracellular vesicles having the PD marker set tetraspanins have at least one tetraspanin such as CD9, CD63, CD81, CD151 on the membrane surface and have at least one tetraspanin selected from CD9, CD63, and CD81. Those having at least one tetraspanin selected from CD9 and CD63 are more preferable, and those having CD9 are particularly preferable.
  • the tetraspanin in the extracellular vesicles having phosphatidylserine and tetraspanin of the PD marker set may be the same as or different from the tetraspanin in the extracellular vesicles having tetraspanin, and the same ones are preferable.
  • the method for assisting the diagnosis of Parkinson's disease in the second invention (hereinafter, may be abbreviated as "PD diagnosis assisting method (II)") is the amount of extracellular vesicles having phosphatidylserine and tetraspanin in a biological sample.
  • the ratio of the amount of phosphatidylserine and extracellular vesicles with tetraspanin to the amount of extracellular vesicles with tetraspanin (PD marker (II))
  • the subject is determined to have Parkinson's disease by using the ratio of the amount of phosphatidylserine and the amount of extracellular vesicles having tetraspanin to the amount of extracellular vesicles having tetraspanin as an index.
  • the biological sample, subject, and extracellular vesicle in the PD diagnostic assist method (II) are the same as those described above, and the preferred ones are also the same.
  • the extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnostic assist method (II) are the same as those described above in the PD marker set, and the preferred ones are also the same.
  • the measurement target of the amount, the amount of extracellular vesicles having phosphatidylserine and tetraspanin, the measurement of the amount of extracellular vesicles having phosphatidylserine and tetraspanin, and the substances used for the measurement are:
  • Examples of the PD diagnostic assisting method include the same as those described above, and an antibody that specifically binds to tetraspanin, and an antibody that specifically binds to tetraspanin and an antibody that specifically binds to phosphatidylserine.
  • preferable ones and specific examples are the same as those described above in the combination of phosphatidylserine-affinitive proteins.
  • Examples of the antibody that specifically binds to tetraspanin in the PD diagnostic assist method (II) include anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody, anti-CD151 antibody, and the like, and anti-CD9 antibody and anti-CD63 antibody.
  • Anti-CD81 antibody is preferable
  • anti-CD9 antibody and anti-CD63 antibody are more preferable
  • anti-CD9 antibody is particularly preferable.
  • As the substance having an affinity for tetraspanin only one kind may be used, or two or more kinds may be used, and it is preferable to use only one kind.
  • the combination of the antibody that specifically binds to tetraspanin and the antibody that specifically binds to phosphatidylserine or the phosphatidylserine-affinitive protein in the PD diagnostic aid method (II) is, for example, specifically to tetraspanin.
  • Examples thereof include a combination of an antibody that binds and an antibody that specifically binds to Tim protein or phosphatidylserine, preferably an anti-CD9 antibody, an anti-CD63 antibody, or a combination of an anti-CD81 antibody and Tim protein, and an anti-CD9 antibody or anti-CD63.
  • the combination of antibody and Tim protein is more preferable, the combination of anti-CD9 antibody or anti-CD63 antibody and Tim1 or Tim4 is more preferable, the combination of anti-CD9 antibody or anti-CD63 antibody and Tim4 is particularly preferable, and the combination of anti-CD9 antibody and Tim4 is particularly preferable. Most preferred.
  • the extracellular vesicles having tetraspanin in the PD diagnostic assist method (II) are the same as those described above in the PD marker set, and the preferred ones are also the same.
  • the tetraspanin in the extracellular vesicle having phosphatidylserine and tetraspanin in the PD diagnostic assist method (II) and the tetraspanin in the extracellular vesicle having tetraspanin may be the same or different, and are the same. Those are preferable.
  • the amount of tetraspanin-bearing extracellular vesicles in the PD diagnostic aid method (II) can be measured by measuring the amount of one type of tetraspanin-bearing extracellular vesicles (for example, only extracellular vesicles having CD9).
  • the amount of two or more types of tetraspanin extracellular vesicles may be measured, and the amount of extracellular vesicles having one type of tetraspanin may be measured. It is preferable to measure only the amount.
  • the measurement of the amount of extracellular vesicles having the tetraspanin is not particularly limited as long as it is a method usually used in this field, and for example, an immunological measurement method using a substance having an affinity for tetraspanin, mass.
  • An analytical method and a method combining these methods can be mentioned, and an immunological measurement method using a substance having an affinity for tetraspanin is preferable.
  • the immunological measurement method includes not only a method using an immune reaction (antigen-antibody reaction) but also a method using a binding force between two molecules other than the antigen-antibody reaction such as binding of a lectin and a protein (a method using the binding force between two molecules.
  • a method similar to the immunological measurement method is also included.
  • Examples of the substance having an affinity for tetraspanin in the measurement of the amount of extracellular vesicles having tetraspanin include the same substances as those described above in the PD diagnostic assist method, and specifically bind to tetraspanin. Proteins are preferred, and antibodies that specifically bind to tetraspanin are more preferred. Examples of the antibody that specifically binds to the tetraspanin include an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, an anti-CD151 antibody, and the like, preferably an anti-CD9 antibody and an anti-CD63 antibody, and an anti-CD9 antibody is preferable. More preferred. As the substance having an affinity for tetraspanin, only one kind may be used, or two or more kinds may be used, and it is preferable to use only one kind.
  • the substance having an affinity for tetraspanin may be a commercially available product or a substance appropriately prepared by a conventional method.
  • the antibody that specifically binds to the tetraspanin may be either a polyclonal antibody or a monoclonal antibody, and these may be used alone or in combination as appropriate.
  • the antibody that specifically binds to the tetraspanin is not only the immunoglobulin molecule itself (intact immunoglobulin), but also a fragment thereof, Fab, F (ab') 2, which has an ability to bind to an antigen.
  • F (ab') and other fragment antibodies may be used.
  • single chain antibodies single chain Fv
  • diabody triabody
  • tetrabody tetrabody and other synthetic antibodies and the like
  • these antibodies may be prepared according to the method described in, for example, "Immunoassay” (edited by Biochemical Measurement Study Group, Kodansha, 2014).
  • the substance having an affinity for tetraspanin in the PD diagnostic assisting method (II) may be labeled with a labeling substance, and the labeling substance and the labeling method are the same as those of the PD diagnostic assisting method and are preferable. And specific examples are the same. Further, as in the PD diagnosis assisting method, a substance having an affinity for tetraspanin is used as a primary affinity substance, and a secondary affinity substance (for example, a secondary antibody) that specifically binds to the primary affinity substance. May be further used.
  • the secondary affinity substance may be labeled with a labeling substance, and the labeling substance and the labeling method are the same as those described above in the PD diagnosis assisting method, and the preferable substances are also the same.
  • labeling with a labeling substance is a substance having an affinity for tetraspanin bound to one of avidins and biotins, and a labeling substance to which the other one of avidins and biotins is bound. May be used to utilize the binding of avidins and biotins.
  • the method for binding avidins and biotins to the avidins, biotins, and substances having an affinity for tetraspanin is the same as that described above in the PD diagnosis assisting method, and the preferred ones and specific examples are also the same. is there.
  • the substance having an affinity for tetraspanin in the PD diagnostic assist method (II) may be immobilized on the solid phase, and the substance having an affinity for the tetraspanin is immobilized on the solid phase or the solid phase.
  • the method for assisting PD diagnosis is the same as that described above, and the preferred method and specific examples are also the same.
  • a substance having an affinity for tetraspanin used for measuring the extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnostic assist method (II) and the extracellular vesicles having the tetraspanin As the substance having an affinity for tetraspanin used, a substance having an affinity for at least one identical tetraspanin (for example, a substance having an affinity for at least CD9 is used for both measurements) is used. However, substances that have an affinity for different types of tetraspanins (eg, substances that have an affinity for CD9 in one measurement and have an affinity for CD63 in the other measurement).
  • (Use) may be used, and it is preferable to use a substance having an affinity for at least one identical tetraspanin, and only a substance having an affinity for the same tetraspanin is used (for example, both). It is more preferable to use only a substance having an affinity for CD9 for the measurement of, and only one kind of substance having an affinity for the same tetraspanin (for example, only an anti-CD9 antibody is used for the measurement of both). It is particularly preferable to use.
  • the measurement and measurement principle of the amount of extracellular vesicles having tetraspanin in the PD diagnostic assist method (II) is the above-mentioned measurement and measurement principle of the amount of extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnostic assist method. And so is the preferred one.
  • the measurement of the amount of tetraspanin-bearing extracellular vesicles in the PD diagnostic assist method (II) specifically includes, for example, (1) affinity for tetraspanin-bearing extracellular vesicles in a biological sample.
  • a method including a step of measuring the amount of the complex (complex amount measuring step) is mentioned as a preferable method.
  • the biological sample is brought into contact with a substance having an affinity for tetraspanin to cause extracellular smallness in the biological sample.
  • the first step of forming a first complex composed of a vesicle and a substance having an affinity for tetraspanin and the contact between the first complex and a substance having an affinity for tetraspanin are brought into contact with each other.
  • the substance having an affinity for tetraspanin used for forming the first complex and the substance having an affinity for tetraspanin used for forming the second complex are the same. Those are preferable.
  • the complex formation step in the measurement of the amount of tetraspanin-bearing extracellular vesicles in the PD diagnostic aid method (II) specifically includes, for example, an antibody that specifically binds to tetraspanin immobilized on a solid phase (an antibody (II).
  • an antibody (II) specifically includes, for example, an antibody that specifically binds to tetraspanin immobilized on a solid phase (an antibody (II).
  • Anti-CD9 antibody, anti-CD63 antibody are preferable, anti-CD9 antibody is more preferable
  • the biological sample are brought into contact with each other to have an antibody that specifically binds to tetraspanin and phosphatidylserine and tetraspanin in the biological sample.
  • a first complex with vesicles is formed, and an antibody that specifically binds to tetraspanin (anti-CD9 antibody, anti-CD63 antibody is preferable, anti-CD9 antibody is more preferable) is brought into contact with the first complex.
  • the first complex is formed with a second complex of an antibody that specifically binds to tetraspanin.
  • washing operation (B / F separation) at least before the complex amount measuring step.
  • the washing operation may be performed after forming the first complex and / or after forming the second complex in the above method, and after forming the first complex. It is preferable to perform the cleaning operation (B / F separation), and further perform the cleaning operation (B / F separation) after forming the second complex.
  • the complex amount measurement step in the measurement of the amount of tetraspanin-containing extracellular vesicles in the PD diagnostic assistance method (II) has an affinity for tetraspanin and the tetraspanin-containing extracellular vesicles obtained in the complex formation step. Any method may be used as long as it is a step of measuring the amount of the complex containing a substance having a property and the amount of the complex can be measured. More specifically, in the complex amount measurement step, for example, (1) an antibody that specifically binds to tetraspanin labeled with a labeling substance is used, or (2) "specifically to tetraspanin".
  • Antibodies (anti-CD9 antibody, anti-CD63 antibody are preferable, anti-CD9 antibody is more preferable) and extracellular vesicles having tetraspanin in biological samples and antibodies that specifically bind to tetraspanin (anti-CD9 antibody, anti-CD63 antibody)
  • the labeling substance in the complex containing the labeling substance enzymes, fluorescent substance is preferable
  • the labeling substance preferably an anti-CD9 antibody
  • the complex amount measurement step it is preferable to perform a washing operation (B / F separation) before detecting the labeling substance.
  • the measurement of the amount of the extracellular vesicles having tetraspanin in the PD diagnostic assist method (II) is more specifically, for example, an antibody (anti-CD9) that specifically binds to tetraspanin immobilized on a solid phase plate.
  • the biological sample is brought into contact with the antibody, which specifically binds to tetraspanin, and the extracellular vesicle having tetraspanin in the biological sample.
  • Antibodies that specifically bind to the first complex and tetraspanin labeled with a labeling substance (anti-CD9 antibody, anti-CD63) after B / F separation, if necessary. Antibodies are preferred, anti-CD9 antibodies are more preferred) to form a second complex of the first complex with an antibody that specifically binds to tetraspanin labeled with a labeling substance.
  • the first complex is brought into contact with an antibody that specifically binds to tetraspanin (anti-CD9 antibody, anti-CD63 antibody is preferable, and anti-CD9 antibody is more preferable), and the first complex is combined with the first complex.
  • a second complex with an antibody that specifically binds to tetraspanin is formed, and if necessary, after B / F separation, the second complex and "an antibody that specifically binds to the tetraspanin"
  • a labeled secondary antibody labeled with a labeling substance, which specifically binds to is contacted to form a third complex of the second complex and the labeled secondary antibody, or (3).
  • a second complex is formed between the first complex and an antibody that specifically binds to tetraspanin to which one of avidins and biotins is bound, and if necessary, after B / F separation, A third complex of a labeling substance (preferably an enzyme or a fluorescent substance) in which the second complex is bound to the remaining one of avidins and biotins is formed, and after B / F separation, it is obtained. It suffices to detect the labeling substance (enzymes, fluorescent substance is preferable) of the 2nd complex or the 3rd complex to be obtained.
  • a labeling substance preferably an enzyme or a fluorescent substance
  • the amount (concentration), labeling substance, labeling method, etc. may be appropriately set according to the type of biological sample, required measurement sensitivity, measurement method to be used, measurement device, and the like.
  • the amount (concentration) of extracellular vesicles having tetraspanin in the biological sample may be calculated by preparing a calibration curve using a standard product. Examples of the standard product include extracellular vesicles having tetraspanin.
  • the ratio of the amount of phosphatidylserine and extracellular vesicles having tetraspanin to the amount of extracellular vesicles having tetraspanin is the amount (A) of extracellular vesicles having phosphatidylserine and tetraspanin, and the cells having tetraspanin. It may be calculated by dividing by the amount of extracellular vesicles (B) ((A) / (B)).
  • the determination of Parkinson's disease in the PD diagnostic assist method (II) is the ratio of the amount (B) of the extracellular vesicles having phosphatidylserine and tetraspanin to the amount (A) of the extracellular vesicles having tetraspanin ((A)). / (B)) is used as an index to determine Parkinson's disease.
  • the determination of Parkinson's disease includes, for example, determination of whether or not the subject is likely to have Parkinson's disease, determination of whether or not the subject is likely to have Parkinson's disease, and determination of whether or not the subject is likely to have Parkinson's disease.
  • Determining whether the subject is at high risk of developing Parkinson's disease determining whether the subject is at risk of developing Parkinson's disease, determining whether the subject is likely to have Parkinson's disease, and determining whether the subject is suffering from Parkinson's disease It is preferable to determine the presence or absence of the possibility of suffering from Parkinson's disease.
  • the ratio of the amount (B) of the extracellular vesicles having phosphatidylserine and tetraspanin to the amount (A) of the extracellular vesicles having tetraspanin ((() Using A) / (B)) as an index, it may include determining (distinguishing) whether the subject has Parkinson's disease with cognitive symptoms or Parkinson's disease without cognitive symptoms.
  • the cognitive symptomatology means a state in which the cognitive function of the brain is reduced due to an acquired organic disorder.
  • Parkinson's disease with cognitive symptoms is defined as having Parkinson's disease or having the possibility of developing Parkinson's disease, and having cognitive symptoms or having the possibility of developing cognitive symptoms. It is a condition, for example, when suffering from Parkinson's disease or when there is a possibility of developing Parkinson's disease and the MMSE is 27 points or less.
  • Parkinson's disease without cognitive symptoms is defined as having Parkinson's disease or having the possibility of developing Parkinson's disease, and having no cognitive symptoms or having the possibility of developing cognitive symptoms. There is no condition, for example, if you have Parkinson's disease or if you are likely to develop Parkinson's disease and your MMSE is greater than 27 points.
  • the determination of Parkinson's disease in the PD diagnostic assist method (II) was obtained, for example, by measuring the amount of extracellular vesicles having tetraspanin described above, and measuring the amount of extracellular vesicles having phosphatidylserine and tetraspanin described above. , The ratio ((A) / (B)) of the amount (B) of phosphatidylserine and extracellular vesicles having tetraspanin to the amount (A) of extracellular vesicles having tetraspanin is set to a predetermined reference value (cut). It is done by comparing with the off value).
  • the ratio ((A) / (B)) of the amount (B) of phosphatidylserine and tetraspanin to the amount of extracellular vesicles having tetraspanin (A) is a predetermined standard. If it is below the value, the subject is likely to have Parkinson's disease, the subject is likely to have Parkinson's disease, or the subject is at high risk of developing Parkinson's disease, or the subject is It can be determined that there is a risk of developing Parkinson's disease.
  • the ratio ((A) / (B)) of the amount (B) of phosphatidylserine and tetraspanin-containing extracellular vesicles to the amount of tetraspanin-containing extracellular vesicles (A) is larger than a predetermined reference value. If the subject is unlikely to have Parkinson's disease, the subject is unlikely to have Parkinson's disease, or the subject is at low risk of developing Parkinson's disease, or the subject is It can be determined that there is no risk of developing Parkinson's disease.
  • the predetermined reference value (cutoff value) in the PD diagnostic assist method (II) is the amount of extracellular vesicles having tetraspanin in the biological sample, which is preset to distinguish between Parkinson's disease patients and healthy subjects.
  • the method for determining the predetermined reference value in the PD diagnostic assist method (II) is not particularly limited, and for example, extracellular vesicles having tetraspanin contained in biological samples obtained from patients suffering from Parkinson's disease and healthy subjects.
  • the amount of extracellular vesicles having phosphatidylserine and tetraspanin relative to the amount of vesicles (A) was measured, and the amount of extracellular vesicles having phosphatidylserine and tetraspanin relative to the amount of extracellular vesicles having tetraspanin (A) was measured.
  • the ratio of the amount (B) ((A) / (B)) was calculated, and the amount of extracellular vesicles having phosphatidylserine and tetraspanin to the amount (A) of the obtained extracellular vesicles having tetraspanin (B).
  • ) ((A) / (B)) can be determined by statistical analysis such as ROC analysis (Receiving Operating Characticanalysis).
  • ROC analysis Receiveiving Operating Characticanalysis
  • the predetermined reference value it is preferable to consider sensitivity, specificity, positive predictive value, negative predictive value, and the like.
  • the predetermined reference value can be set so that the sensitivity is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, and 90% or more. More preferably, for example, the specificity can be determined to be 60% or more, 70% or more is preferable, 80% or more is more preferable, and 90% or more is further preferable.
  • the determination of Parkinson's disease in the PD diagnosis assisting method (II) is based on the ratio of the amount (B) of the extracellular vesicles having phosphatidylserine and tetraspanin to the amount (A) of the extracellular vesicles having tetraspanin ((A)). / (B)) is used as an index to determine whether the subject is a healthy person, has Parkinson's disease with cognitive symptoms, or has Parkinson's disease without cognitive symptoms.
  • a cut-off value for distinguishing between healthy people and Parkinson's disease with cognitive symptoms (hereinafter sometimes abbreviated as "healthy person / Parkinson's disease standard value with cognitive symptoms”) and Parkinson's disease with cognitive symptoms Judgment (distingment) may be made based on a cut-off value for distinguishing Parkinson's disease without cognitive symptoms (hereinafter sometimes abbreviated as "Parkinson's disease with cognitive symptoms / Parkinson's disease standard value without cognitive symptoms") ⁇ , "Parkinson's disease with cognitive symptoms / Parkinson's disease without cognitive symptoms” is used to determine (distinguish) only whether the subject has Parkinson's disease with cognitive symptoms or Parkinson's disease without cognitive symptoms. You may.
  • the subject when the subject only determines whether the subject has Parkinson's disease with cognitive symptoms or Parkinson's disease without cognitive symptoms, the subject is diagnosed with Parkinson's disease based on the diagnostic criteria. People who have been diagnosed with Parkinson's disease based on diagnostic criteria are preferred.
  • the diagnostic criteria are the same as those described above.
  • the determination of whether the subject has Parkinson's disease with cognitive symptoms or Parkinson's disease without cognitive symptoms is determined, for example, by the amount of extracellular vesicles having tetraspanin described above.
  • the amount of extracellular vesicles having phosphatidylserine and tetraspanin relative to the amount (A) of the extracellular vesicles having tetraspanin obtained by the measurement and the measurement of the amount of extracellular vesicles having phosphatidylserine and tetraspanin described above It is done by comparing the ratio of B) ((A) / (B)) with a predetermined “Parkinson's disease with cognitive symptom / Parkinson's disease standard value without cognitive symptom”.
  • the ratio ((A) / (B)) of the amount (B) of phosphatidylserine and tetraspanin-containing extracellular vesicles to the amount of tetraspanin-containing extracellular vesicles (A) is "cognitive symptoms. If a Parkinson's disease / Parkinson's disease without cognitive symptoms is below the standard value, the subject is likely to have Parkinson's disease without cognitive symptoms, or the subject is at risk of developing Parkinson's disease without cognitive symptoms. It can be determined that the subject is at high risk of developing Parkinson's disease without cognitive symptoms.
  • the ratio ((A) / (B)) of the amount (B) of phosphatidylserine and tetraspanin-containing extracellular vesicles to the amount of tetraspanin-containing extracellular vesicles (A) is "Parkinson's disease with cognitive symptoms. If it is larger than / Parkinson's disease without cognitive symptoms, the subject is likely to have Parkinson's disease with cognitive symptoms, or the subject is at high risk of developing Parkinson's disease with cognitive symptoms. Alternatively, it can be determined that the subject is at risk of developing Parkinson's disease with cognitive symptoms.
  • the method for determining "Parkinson's disease with cognitive symptoms / Criteria for Parkinson's disease without cognitive symptoms” is not particularly limited, and for example, patients suffering from Parkinson's disease with cognitive symptoms and Parkinson's disease without cognitive symptoms.
  • the amount of phosphatidylserine and tetraspanin-containing extracellular vesicles (B) was measured with respect to the amount of tetraspanin-containing extracellular vesicles (A) contained in the biological sample obtained from the patient, and the amount of tetraspanin-containing extracellular vesicles (B) was measured.
  • the ratio ((A) / (B)) of the amount of extracellular vesicles (B) having phosphatidylserine and tetraspanin to the amount of vesicles (A) was calculated, and the amount of extracellular vesicles having tetraspanin obtained was calculated.
  • ROC analysis Receiveiver Operating Characticanalysis
  • the predetermined reference value can be set so that the sensitivity is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, and 90% or more. More preferably, for example, the specificity can be determined to be 60% or more, 70% or more is preferable, 80% or more is more preferable, and 90% or more is further preferable.
  • the determination of whether the subject in the PD diagnostic assistance method (II) is a healthy person, Parkinson's disease with cognitive symptoms, or Parkinson's disease without cognitive symptoms is determined, for example, of the extracellular vesicle having tetraspanin.
  • the ratio ((A) / (B)) of the amount (B) of the extracellular vesicles having phosphatidylserine and tetraspanin to the amount (A) is "Parkinson's disease with cognitive symptoms / Parkinson's disease without cognitive symptoms reference value". If the following are the following, the subject is likely to have Parkinson's disease without cognitive symptoms, or the subject is at high risk of developing Parkinson's disease without cognitive symptoms, or the subject has Parkinson's disease without cognitive symptoms.
  • the subject At risk of developing; if the subject has cognitive symptoms greater than "Parkinson's disease with cognitive symptoms / Parkinson's disease without cognitive symptoms” and less than or less than "healthy individuals / Parkinson's disease with cognitive symptoms” Highly likely to have Parkinson's disease, or subjects at high risk of developing cognitive Parkinson's disease, or subjects at high risk of developing cognitive Parkinson's disease; "healthy / cognitive symptoms” If it is larger than "the standard value for Parkinson's disease", the subject is unlikely to have Parkinson's disease, the subject is unlikely to have Parkinson's disease, or the subject has Parkinson's disease. It can be determined that the risk of developing Parkinson's disease is low, or that the subject is not at risk of developing Parkinson's disease.
  • the method for determining the "healthy subject / Parkinson's disease reference value with cognitive symptoms” is not particularly limited, and for example, extracellular vesicles having tetraspanin contained in biological samples obtained from healthy subjects and Parkinson's disease patients with cognitive symptoms.
  • the amount of extracellular vesicles having phosphatidylserine and tetraspanin relative to the amount (A) of (A) was measured, and the amount of extracellular vesicles having phosphatidylserine and tetraspanin relative to the amount of extracellular vesicles having tetraspanin (A).
  • the ratio of (B) ((A) / (B)) was calculated, respectively, and the amount of phosphatidylserine and tetraspanin-containing extracellular vesicles (B) with respect to the obtained amount of tetraspanin-containing extracellular vesicles (A).
  • ((A) / (B)) can be determined by statistical analysis such as ROC analysis (Receiving Operating Characticanalysis).
  • ROC analysis Receiveiving Operating Characticanalysis
  • the predetermined reference value it is preferable to consider sensitivity, specificity, positive predictive value, negative predictive value, and the like.
  • the predetermined reference value can be set so that the sensitivity is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, and 90% or more. More preferably, for example, the specificity can be determined to be 60% or more, 70% or more is preferable, 80% or more is more preferable, and 90% or more is further preferable.
  • the reagent kit for assisting diagnosis of Parkinson's disease in the second invention (hereinafter, may be abbreviated as "reagent kit for assisting PD diagnosis (II)") is for a substance having an affinity for tetraspanin and phosphatidylserine. Includes substances that have an affinity for
  • the substances having an affinity for tetraspanin and the substances having an affinity for phosphatidylserine in the PD diagnostic assisting reagent kit (II) are the same as those described above in the PD diagnostic assisting method (II), and are preferable. The same is true for things.
  • the substance having an affinity for the tetraspanin and the substance having an affinity for the phosphatidylserine may be in a solution state, a frozen state, a dried state, or a freeze-dried state, respectively. Further, the substance having an affinity for the tetraspanin and the substance having an affinity for the phosphatidylserine may be included in the kit as one reagent or included in the kit as separate reagents. ..
  • the combination of the substance having an affinity for tetraspanin and the substance having an affinity for phosphatidylserine in the PD diagnostic assisting reagent kit (II) is the same as the combination described above in the PD diagnostic assisting method (II). The same applies to the preferred combinations.
  • the substance having an affinity for tetraspanin and / and the substance having an affinity for phosphatidylserine in the PD diagnostic aid reagent kit (II) may be immobilized on a solid phase and labeled with a labeling substance. It may have been done.
  • the solid phase, the method for immobilizing the solid phase, the labeling substance, and the labeling method are the same as those described above in the PD diagnosis assisting method, and the preferred ones are also the same.
  • the PD diagnostic aid reagent kit (II) is a secondary affinity substance (eg,) that specifically binds to a substance having an affinity for the tetraspanin and / or a substance having an affinity for the phosphatidylserine. Secondary antibody) may be further included.
  • the secondary affinity substance in the PD diagnostic assisting reagent kit (II) is the same as that described above in the PD diagnostic assisting method, and the preferred one is also the same.
  • the secondary affinity substance in the PD diagnosis assisting reagent kit (II) may be labeled with a labeling substance, and the labeling substance and the labeling method are the same as those described above in the PD diagnosis assisting method. Yes, and the preferred ones are the same.
  • the substance having an affinity for tetraspanin and / and the substance having an affinity for phosphatidylserine in the PD diagnostic aid reagent kit (II) may be a substance in which one of avidins and biotins is bound, and the avidin.
  • the types and biotins are the same as those described above in the PD diagnosis assisting method, and the preferred ones are also the same.
  • the PD diagnostic assisting reagent kit (II) may contain a labeling substance to which one of avidins and biotins is bound, and the labeling substance and labeling method are the same as those described above in the PD diagnostic assisting method. The same applies to the preferred ones.
  • the concentration (amount) of the substance having an affinity for tetraspanin and the substance having an affinity for phosphatidylserine in the PD diagnostic aid reagent kit (II) is usually used in this field depending on the measurement method. It may be set appropriately within the range.
  • the substance having an affinity for tetraspanin preferably contains, for example, a concentration at the time of use, which is usually 10 to 20000 ng / mL or 100 to 10000 ng / mL when immobilized on a solid phase. When used for detection, those containing an amount of 10 to 5000 ng / mL and 100 to 500 ng / mL are usually preferable.
  • the substance having an affinity for phosphatidylserine contains, for example, a concentration at the time of use, which is usually 10 to 20000 ng / mL or 100 to 10000 ng / mL when immobilized on a solid phase. Is preferable, and when it is used for detection, it is usually preferably one containing an amount of 10 to 5000 ng / mL and 100 to 500 ng / mL.
  • the substance having an affinity for tetraspanin and the substance having an affinity for phosphatidylserine in the reagent kit (II) for assisting PD diagnosis may coexist with reagents usually used in this field.
  • the reagent is the same as the reagent kit for PD diagnosis assistance.
  • the PD diagnostic aid reagent kit (II) includes substances having an affinity for tetraspanin and substances having an affinity for phosphatidylserine, as well as tetraspanin and phosphatidylserine using these affinity substances.
  • the reagents necessary for measuring the amount of extracellular vesicles having and / and the amount of extracellular vesicles having phosphatidylserine may be provided.
  • Such a reagent is the same as that of the PD diagnosis assisting reagent kit.
  • the PD diagnostic aid reagent kit (II) may include a standard product used to prepare a calibration curve for extracellular vesicles having phosphatidylserine and tetraspanin, and the standard product is for PD diagnostic aid. Similar to the reagent kit.
  • the PD diagnostic aid reagent kit (II) may contain a standard product used for preparing a calibration curve for extracellular vesicles having tetraspanin, and the standard product includes extracellular vesicles having tetraspanin. Vesicles are mentioned.
  • the standard product may be in a solution state, a frozen state, a dried state, or a freeze-dried state.
  • reagents usually used in this field such as buffers, reaction accelerators, sugars, proteins, salts, stabilizers such as surfactants, preservatives, etc.
  • concentrations and pHs may be appropriately selected from the ranges usually used in this field.
  • the PD diagnostic aid reagent kit (II) may include package inserts and instruction manuals.
  • the attached documents and instruction manuals include, for example, measuring the amount of extracellular vesicles having tetraspanin and the amount of extracellular vesicles having phosphatidylserine and tetraspanin in a biological sample derived from a subject, and / and. Using the ratio of the amount of phosphatidylserine and the amount of extracellular vesicles having tetraspanin to the amount of extracellular vesicles having tetraspanin as an index, it is possible to determine that the subject has Parkinson's disease (for example, PD diagnostic assist method (II)). Examples include the attached documents and instruction manuals described. These package inserts and instruction manuals may be described separately in a plurality of cases, or may be described collectively in one case.
  • the remaining one of the substances having an affinity is bound to the labeling substance, or the other affinity substance and the component (B) that indirectly binds the affinity to the labeling substance have an affinity for tetraspanin.
  • kits (II) for example, a kit containing the following (C) and (D) is preferable.
  • C) One of a substance having an affinity for tetraspanin and a substance having an affinity for phosphatidylserine immobilized on a solid phase, and one selected from the following (1) to (3).
  • One of the substances having an affinity for phosphatidylserine and one of the substances having an affinity for phosphatidylserine is bound to one of avidins and biotins, and the other of avidins and biotins is bound to a labeling substance (D).
  • a substance having an affinity for tetraspanin is a labeling substance.
  • Preferred examples of the PD diagnostic assist reagent kit (II) include the following.
  • a kit containing a solid phase plate on which an anti-CD9 antibody is particularly preferable) and any one selected from the following (1) to (3) can be mentioned.
  • a solution containing a tetraspanin antibody (anti-CD9 antibody or anti-CD63 antibody is more preferable, anti-CD9 antibody is particularly preferable) bound to a labeling substance (usually 10 to 5000 ng / mL, 100 to 500 ng / mL is preferable).
  • a labeling substance usually 10 to 5000 ng / mL, 100 to 500 ng / mL is preferable.
  • a solution containing a tetraspanin antibody (anti-CD9 antibody or anti-CD63 antibody is more preferable, anti-CD9 antibody is particularly preferable) (usually 10 to 5000 ng / mL, 100 to 500 ng / mL is preferable), and the tetraspanin antibody.
  • a solution containing a secondary affinity substance specifically bound to the labeling substance (usually 10 to 5000 ng / mL, preferably 100 to 500 ng / mL), (3) one of avidins and biotins
  • a solution (usually 10-5000 ng / mL, preferably 100-500 ng / mL) containing an antibody that specifically binds to bound tetraspanin (preferably anti-CD9 antibody or anti-CD63 antibody, more preferably anti-CD9 antibody).
  • a solution containing a labeling substance to which the other one of avidins and biotins is bound (usually 10 to 5000 ng / mL, preferably 100 to 500 ng / mL).
  • the device for assisting the diagnosis of Parkinson's disease in the second invention (hereinafter, may be abbreviated as "device for assisting the diagnosis of PD (II)”) is an extracellular sample having phosphatidylserine and tetraspanin in a biological sample derived from a subject.
  • a measuring unit that measures the amount of vesicles and the amount of extracellular vesicles having tetraspanin, and the ratio of the amount of extracellular vesicles having phosphatidylserine and tetraspanin to the amount of extracellular vesicles having tetraspanin.
  • the subject has Parkinson's disease using the calculation unit and the ratio of the amount of phosphatidylserine and tetraspanin-containing extracellular vesicles in the biological sample to the amount of tetraspanin-containing extracellular vesicles in the biological sample as an index. It has a determination unit for determining.
  • the measurement unit, calculation unit, determination unit, etc. constituting the PD diagnosis assisting device (II) may be arranged in the same device or may be separate from each other.
  • the measuring unit in the PD diagnostic assisting device (II) is a site for measuring the amount of extracellular vesicles having phosphatidylserine and tetraspanin and the amount of extracellular vesicles having tetraspanin in the biological sample introduced into the device. Is.
  • the size and configuration of the measuring unit are not particularly limited.
  • the measuring unit is, for example, an imaging device used for imaging using a microplate reader or CCD used in an ELISA method, an imaging device used in a method using a Western blotting method or a microarray (microchip), and a mass spectrometry method. Examples thereof include mass spectrometers, intermolecular interaction analyzers, flow cytometers used for flow cytometry, ultraviolet visible light detectors and fluorescence detectors used for HPLC methods and capillary electrophoresis methods.
  • the subjects, biological samples, and extracellular vesicles in the PD diagnostic assisting device (II) are the same as those described above, and the preferred ones are also the same.
  • the measurement target of the amount of extracellular vesicles having phosphatidylserine and tetraspanin and the amount of extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnostic assisting device (II) is the same as that described above in the PD diagnostic assisting method. Yes, and the preferred ones are the same.
  • the measurement of the amount of extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnosis assisting device (II) and the substances used for the measurement are the same as those described above in the PD diagnosis assisting method, and are preferable or specific. The example is similar.
  • the measurement target of the amount of the extracellular vesicle having tetraspanin and the extracellular vesicle having tetraspanin in the PD diagnosis assisting device (II) is the same as that described above in the PD diagnosis assisting method (II), which is preferable. The same is true for things.
  • the measurement of the amount of tetraspanin-containing extracellular vesicles in the PD diagnostic assisting device (II) and the substances used for the measurement are the same as those described above in the PD diagnostic assisting method (II), and preferred ones and specific examples. Is the same.
  • the calculation unit in the PD diagnostic assist device (II) is based on the amount of extracellular vesicles having tetraspanin and the amount of extracellular vesicles having phosphatidylserine and tetraspanin obtained in the measuring unit, and the extracellular vesicles having tetraspanin. It is a site for calculating the ratio of the amount of extracellular vesicles having phosphatidylserine and tetraspanin to the amount of vesicles.
  • the size and configuration of the measuring unit are not particularly limited.
  • the calculation unit in the PD diagnostic assisting device (II) is the mass of the extracellular vesicles having phosphatidylserine and tetraspanin and the extracellular vesicles having tetraspanin in the biological sample obtained by the measuring unit in the PD diagnostic assisting device.
  • Measured values that correlate with the concentration and concentration for example, absorbance, change in absorbance, transmitted light, change in transmitted light, fluorescence intensity, change in fluorescence intensity, emission amount, change in emission amount, turbidity, turbidity change rate, After converting scattered light, scattered light change rate, reflectance, reflectance change amount, refractive index, refractive index change amount, etc.) into mass, concentration, etc., phosphatidylserine and tetraspanin with respect to the amount of extracellular vesicles having tetraspanin. You may calculate the proportion of the amount of extracellular vesicles having.
  • the ratio of the amount of phosphatidylserine and the amount of extracellular vesicles having tetraspanin to the amount of the extracellular vesicles having tetraspanin, such as the measured value and the mass and concentration converted by the calculation unit, is determined in a device such as a memory or a hard disk. It may be stored in a provided storage device or the like.
  • the determination unit in the PD diagnostic assisting device (II) determines Parkinson's disease using the ratio of the amount of phosphatidylserine and the amount of extracellular vesicles having tetraspanin to the amount of extracellular vesicles having tetraspanin calculated by the calculation unit as an index. It is a part to be used. Further, the determination unit in the PD diagnosis assisting device (II) may be a site for distinguishing whether the subject has Parkinson's disease with cognitive symptoms or Parkinson's disease without cognitive symptoms.
  • the determination of Parkinson's disease in the PD diagnosis assisting device (II), the predetermined reference value used for the determination, and the determination method of the predetermined reference value are the same as those described above in the PD diagnosis assisting method (II). The same applies to preferable ones and specific examples.
  • the predetermined reference value in the PD diagnosis assisting device (II) may be stored in advance in the PD diagnosis assisting device (II), or may be stored in advance from the input site of the PD diagnosis assisting device (II) at the time of determination. It may be entered.
  • the PD diagnosis assisting device (II) may include an output unit.
  • the output unit performs processing such as displaying or outputting the result of the determination on a display device such as a display or a printing device such as a printer.
  • data (determination result) for assisting a doctor's diagnosis of Parkinson's disease can be obtained.
  • the doctor relates to, for example, a biomarker for Parkinson's disease recommended in the Parkinson's disease medical treatment guidelines such as interview, MIBG myocardial scintigraphy, and dopamine transporter scintigraphy.
  • the test and the test using the candidate substance of the biomarker of Parkinson's disease may be further performed, and the subject can make a diagnosis of Parkinson's disease in consideration of these results and the like.
  • a drug for Parkinson's disease drug or therapeutic drug for delaying the progression of Parkinson's disease
  • a drug for Parkinson's disease drug or therapeutic drug for delaying the progression of Parkinson's disease
  • L-dopa levodopa
  • a drug that may adversely affect cognitive symptoms such as an anticholinergic drug, and for example, dementia such as a cholinesterase inhibitor or mild cognitive impairment. It is preferable to administer drugs for cognitive impairment (drugs and therapeutic agents that slow the progression of dementia and mild cognitive impairment) and perform surgery.
  • determining (distinguishing) whether a subject has Parkinson's disease without cognitive symptoms or Parkinson's disease with cognitive symptoms is useful for a doctor to determine a treatment policy or make a diagnosis.
  • the method, biomarker, reagent kit and device for assisting the diagnosis of Parkinson's disease of the present invention are useful in the field of clinical examination because they can assist the diagnosis of Parkinson's disease. According to the present invention, it is possible to assist in the diagnosis of Parkinson's disease with high accuracy.
  • Example 1 Evaluation of Parkinson's disease specimens (MMSE greater than 27) using the amount of exosomes having PS and CD9 as an index
  • the exosomes were measured by the sandwich ELISA method of Tim protein-anti-CD9 antibody, and the AUC and p values were calculated.
  • MagCapture registered trademark
  • Exosome Solution Kit PS manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter referred to as "A kit”
  • the protein concentration in the obtained exosome solution was measured by the BCA method (bicinchoninic acid method).
  • the obtained exosome solution was measured by the BCA method using the Reaction Buffer attached to PS Capture Exosome ELISA Kit, Streptavidin HRP (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter referred to as "B kit").
  • the reagents attached to the B kit were used.
  • the Washing Buffer (10 ⁇ ) attached to the B kit was diluted 10-fold with purified water (distilled water), and then the Exosome Binding Enhancer (100 ⁇ ) attached to the B kit was obtained with respect to the diluted solution. 1/100 amount was added. The obtained solution is referred to as "cleaning solution (1x)".
  • cleaning solution (1x) the Exosome Binding Enhancer
  • the reaction solution was discarded, and each well was washed 3 times with 300 to 350 ⁇ L of the washing solution ( ⁇ 1). Then, using the Reaction Buffer included in the B kit, the biotin-labeled anti-CD9 mouse monoclonal antibody was diluted to a final concentration of 250 ng / mL to obtain a biotin-labeled antibody reaction solution. 100 ⁇ L of the obtained biotin-labeled antibody reaction solution was dispensed into each well, a plate seal was attached, and the reaction was carried out at room temperature for 1 hour while stirring at about 500 rpm using a microplate shaker.
  • reaction solution was discarded, and each well was washed 3 times with 300 to 350 ⁇ L of the washing solution ( ⁇ 1).
  • Reaction Buffer included in the B kit 1/100 amount of HRP-conjuged Streptavidin (100 ⁇ ) was added and mixed well to prepare an HRP-labeled streptavidin reaction solution (1 ⁇ ).
  • 100 ⁇ L of the obtained HRP-labeled streptavidin reaction solution (1 ⁇ ) was dispensed into each well, a plate seal was attached, and the mixture was reacted at room temperature for 2 hours while stirring at about 500 rpm using a microplate shaker.
  • reaction solution was discarded, and each well was washed 5 times with 300 to 350 ⁇ L of the washing solution (1 ⁇ ).
  • 100 ⁇ L of the TMB (3,3', 5,5'-tetramethylbenzidine) Solution attached to the B kit returned to room temperature was dispensed into each well, and the mixture was stirred for about 1 minute using a microplate shaker. After that, a plate seal was attached and the mixture was allowed to stand for 30 minutes at room temperature (20 to 25 ° C.). Then, 100 ⁇ L of Stop Solution attached to the B kit returned to room temperature was added to each well, and after stirring for about 5 seconds using a microplate shaker, a 96-well microplate reader (Tecan, Safari2) was immediately used.
  • the absorbance at 450 nm and the absorbance at the sub-wavelength of 620 nm were measured using the mixture.
  • the value obtained by subtracting the sub-wavelength 620 nm absorbance value from the 450 nm absorbance value was defined as the "absorbance value”, and the value obtained by subtracting the blank absorbance value from the absorbance value of the sample diluent for measurement was calculated as the "corrected sample absorbance value”. ..
  • a standard curve was created from the value obtained by subtracting the blank absorbance value from the absorbance value of the exosome dilution series (calibrator) derived from the COLO201 cell culture supernatant and the protein concentration of the calibrator.
  • the corrected sample absorbance value was converted into a protein concentration using a standard curve, and the value obtained by multiplying the obtained conversion value by the sample dilution rate was taken as the “sample measurement value” [ng / mL].
  • (4) Calculation of AUC Based on the sample measurement value obtained in (3), Wilcoxon / Kruskal-Wallis test (registered trademark) 11 (SAS Institute Inc., Carry, NC, USA) was used. The p-value was calculated by performing a significant difference test between Parkinson's disease patients and healthy subjects. Further, based on the sample measurement value obtained in (3), a logistic regression analysis was performed using JMP (registered trademark) 11 (SAS Institute Inc., Cary, NC, USA), and the obtained receiver operating characteristics were obtained.
  • JMP registered trademark
  • the area under the curve (AUC) was calculated from the curve (ROC curve). The obtained results are shown in Table 1 below.
  • a box-and-whisker graph created based on the sample measurement values is shown in FIG. In the figure, the vertical axis shows the sample measurement value, the horizontal axis shows the result of a healthy person, and PD shows the result of a Parkinson's disease patient (MMSE is larger than 27).
  • Comparative example 1 Evaluation of Parkinson's disease specimen (MMSE greater than 27) using the amount of exosomes having CD9 as an index Anti-CD9 antibody was immobilized on the solid phase in place of Tim4 protein, and the same method as in Example 1 was used. Exosomes were measured by the CD9 antibody-anti-CD9 antibody sandwich ELISA method, and a significant difference test was performed between Parkinson's disease patients (MMSE greater than 27) and healthy subjects, and AUC and p values were calculated. A plate on which the anti-CD9 antibody was immobilized on a solid phase was prepared by the following method.
  • Anti-CD9 mouse monoclonal antibody (1K) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted with 50 mM MOPS (pH 7.5) to a concentration of 10 ⁇ g / mL, and 100 ⁇ L was added to the wells of a 96-well microplate (Nunc). Then, it was incubated overnight in a refrigerator. Wells were washed 3 times with TBST (Tris Buffered Saline, pH 7.4), 300 ⁇ L of TBS (Tris Buffered Saline, pH 7.4) containing 10 mg / mL block ace was added, and the cells were incubated overnight in a refrigerator and antibody-solidified. Used as a companion plate.
  • TBST Tris Buffered Saline, pH 7.4
  • TBS Tris Buffered Saline, pH 7.4
  • Example 2 As the anti-CD9 antibody of the detection antibody, a biotin-labeled antibody was used in the same manner as in Example 1. The obtained results are shown in Table 1 below.
  • a box-and-whisker graph created based on the sample measurement values is shown in FIG. In the figure, the vertical axis shows the sample measurement value, the horizontal axis shows the result of a healthy person, and PD shows the result of a Parkinson's disease patient (MMSE is larger than 27).
  • Example 2 Evaluation of Parkinson's disease sample (MMSE is greater than 27) using the amount of exosomes having PS and CD63 as an index
  • Tim protein-anti-CD63 antibody sandwich ELISA method was used to measure exosomes, and a significant difference test was performed between Parkinson's disease patients (MMSE greater than 27) and healthy subjects to calculate AUC and p values.
  • the anti-CD63 antibody of the detection antibody the biotin-labeled anti-CD63 antibody attached to PS Capture Exosome ELISA Kit, Streptavidin HRP (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., the above “B kit”) was used. The obtained results are shown in Table 1 below.
  • Comparative example 2 Evaluation of Parkinson's disease specimen using CD63 as an index Comparison except that anti-CD63 mouse monoclonal antibody (3-13) (Fujifilm Wako Pure Chemical Industries, Ltd.) was immobilized on the solid phase instead of anti-CD9 antibody and used as a detection antibody. Exosomes were measured by the anti-CD63 antibody-anti-CD63 antibody sandwich ELISA method in the same manner as in Example 1, and a significant difference test was performed between Parkinson's disease patients (MMSE greater than 27) and healthy subjects, and the AUC and p values were determined. Calculated. A plate on which the anti-CD63 antibody was immobilized on a solid phase was prepared by the same method as in Comparative Example 1.
  • the anti-CD63 antibody of the detection antibody As the anti-CD63 antibody of the detection antibody, the biotin-labeled anti-CD63 antibody attached to PS Capture Exosome ELISA Kit, Streptavidin HRP (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., the above “B kit”) was used. The obtained results are shown in Table 1 below.
  • Example 3 Evaluation of Parkinson's Disease Dementia Specimen Using the Amount of Exosomes Having PS and CD81 as an Index
  • An anti-CD81 mouse monoclonal antibody (17B1) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used instead of the anti-CD9 antibody as a detection antibody.
  • Exosomes were measured by the sandwich ELISA method of Tim protein-anti-CD81 antibody by the same method as in Example 1, and a significant difference test was performed between Parkinson's disease patients (MMSE greater than 27) and healthy subjects. The p value was calculated.
  • As the anti-CD81 antibody of the detection antibody a biotin-labeled antibody was used in the same manner as in Example 1. The obtained results are shown in Table 1 below.
  • Example 4 Evaluation of Parkinson's disease sample (MMSE is greater than 27) using the ratio of the amount of PS and the amount of exosomes having CD9 to the amount of exosomes having CD9 Obtained in Example 1 (Tim protein-anti-CD9 antibody sandwich ELISA method). Divide the obtained “sample measurement value” (value (A)) by the “sample measurement value” (value (B)) obtained in Comparative Example 1 (anti-CD9 antibody-anti-CD9 antibody sandwich ELISA method). (A) / (B) (hereinafter referred to as "corrected sample measurement value”) was obtained.
  • FIG. 2 shows a box-and-whisker graph created based on the corrected sample measurement values.
  • the vertical axis shows the adjusted sample measurement values ((A) / (B))
  • the horizontal axis shows the results of healthy subjects
  • PD shows the results of Parkinson's disease patients (MMSE is larger than 27).
  • Example 5 Evaluation of Parkinson's disease sample (MMSE is greater than 27) using the ratio of the amount of PS and the amount of exosomes having CD63 to the amount of exosomes having CD63 Obtained in Example 2 (Tim protein-anti-CD63 antibody sandwich ELISA method). Divide the obtained "sample measurement value" (value (A)) by the “sample measurement value” (value (B)) obtained in Comparative Example 2 (anti-CD63 antibody-anti-CD63 antibody sandwich ELISA method). (A) / (B) (corrected sample measurement value) was obtained.
  • Parkinson's disease was evaluated using plasma from patients with Parkinson's disease (MMSE greater than 27) without cognitive symptoms and plasma from healthy subjects, respectively, and the amount of tetraspanin-bearing exosomes of CD9, CD63, or CD81 as an index.
  • the AUC was 0.855, 0.848, and 0.642, respectively.
  • the amount of exosomes having phosphatidylserine to which tetraspanin and Tim protein of CD9, CD63, or CD81 bind is used as an index.
  • the AUCs were 0.929, 0.899, and 0.916, respectively, and it was found that Parkinson's disease (particularly Parkinson's disease without cognitive symptoms) can be detected with high accuracy.
  • the corrected sample measurement value ((value (B)) obtained by dividing the sample measurement value (value (A)) of the exosome having phosphatidylserine and CD63 by the sample measurement value (value (B)) of the exosome having CD63.
  • the AUC was 0.892, and it was found that Parkinson's disease (particularly Parkinson's disease without cognitive symptoms) could be detected with high accuracy.
  • the corrected sample measurement value ((value (B)) obtained by dividing the sample measurement value (value (A)) of the exosome having phosphatidylserine and CD9 by the sample measurement value (value (B)) of the exosome having CD9.
  • the AUC was 0.973, and it was found that Parkinson's disease (particularly Parkinson's disease without cognitive symptoms) can be detected with extremely high accuracy.
  • Example 6 Evaluation of Parkinson's disease specimens (MMSE 27 or less) using the ratio of the amount of PS and CD9 exosomes to the amount of CD9 exosomes "8 Parkinson's disease patients (MMSE 27 or less) purchased from PrecisionMed", Exosomes were measured by the sandwich ELISA method of Tim tan parkin-anti-CD9 antibody by the same method as in Example 1 except that "EDTA plasma of 30 healthy subjects” was used, and "sample measurement value” (sample measurement value) Value (A)) was obtained. In addition, anti-CD9 antibody-anti-CD9 antibody by the same method as in Comparative Example 1 except that "8 samples of Parkinson's disease patients (MMSE of 27 or less) and 30 samples of healthy subjects purchased from PrecisionMed" were used.
  • FIG. 3 shows a box-and-whisker graph created based on the corrected sample measurement values.
  • the vertical axis is the sample measurement value
  • the horizontal axis is the result of healthy subjects
  • PD MMSE is 27 or less
  • PD MMSE is greater than 27
  • the results are shown respectively.
  • Example 7 Evaluation of Parkinson's disease specimens (MMSE greater than 27) using the amount of exosomes with PS and CD9 as an index "8 Parkinson's disease patients (MMSE greater than 27) purchased from PrecisionMed, 30 healthy subjects with EDTA plasma Exosomes were measured by the sandwich ELISA method of Tim protein-anti-CD9 antibody by the same method as in Example 1, and a significant difference between Parkinson's disease patients (MMSE greater than 27) and healthy subjects. The test was performed and the AUC and p values were calculated. The obtained results are shown in Table 3 below.
  • Comparative example 4 Evaluation of Parkinson's disease specimens (MMSE greater than 27) using the amount of exosomes having CD9 as an index "8 Parkinson's disease specimens (MMSE greater than 27) purchased from PrecisionMed, 30 healthy subjects of EDTA plasma" Exosomes were measured by the anti-CD9 antibody-anti-CD9 antibody sandwich ELISA method in the same manner as in Comparative Example 1 except that they were used, and a significant difference test between Parkinson's disease patients (MMSE greater than 27) and healthy subjects was tested. Was performed, and the AUC and p values were calculated. The obtained results are shown in Table 3 below.
  • Example 8 Evaluation of Parkinson's disease sample (MMSE greater than 27) using the ratio of the amount of PS and the amount of exosomes having CD9 to the amount of exosomes having CD9 Obtained in Example 7 (Tim protein-anti-CD9 antibody sandwich ELISA method) Divide the obtained "sample measurement value" (value (A)) by the “sample measurement value” (value (B)) obtained in Comparative Example 4 (anti-CD9 antibody-anti-CD9 antibody sandwich ELISA method). (A) / (B) (hereinafter referred to as "corrected sample measurement value”) was obtained.
  • FIG. 3 shows a box-and-whisker graph created based on the corrected sample measurement values.
  • Example 9 Evaluation of Parkinson's disease specimens (MMSE is 27 or less) and Parkinson's disease specimens (MMSE is greater than 27) using the ratio of the amount of PS and the amount of exosomes having CD9 to the amount of exosomes having CD9 "Parkinson purchased from PrecisionMed" Tim protein-anti-CD9 by the same method as in Example 1 except that 8 samples of diseased patients (MMSE of 27 or less) and 8 samples of Parkinson's disease patients (MMSE of greater than 27) purchased from PrecisionMed were used. Exosomes were measured by the antibody sandwich ELISA method to obtain "sample measurement values" (values (A)).
  • FIG. 3 shows a box-and-whisker graph created based on the corrected sample measurement values.
  • Example 10 Evaluation of Parkinson's disease specimens using the amount of exosomes having PS and CD9 as an index "16 Parkinson's disease patients purchased from PrecisionMed (8 specimens with MMSE of 27 or less and 8 specimens with MMSE greater than 27), 30 healthy subjects Exosomes were measured by the sandwich ELISA method of Tim protein-anti-CD9 antibody by the same method as in Example 1 except that "EDTA plasma” was used, and a significant difference test between Parkinson's disease patients and healthy subjects was performed. And the p value were calculated. The obtained results are shown in Table 5 below.
  • a box-and-whisker graph created based on the sample measurement values is shown in FIG. In the figure, the vertical axis shows the sample measurement value, the horizontal axis shows the result of a healthy person, and PD shows the result of a Parkinson's disease patient.
  • Comparative example 7 Evaluation of Parkinson's disease specimens using the amount of exosomes having CD9 as an index "16 Parkinson's disease patients purchased from PrecisionMed (8 specimens with MMSE of 27 or less and 8 specimens with MMSE greater than 27), 30 healthy subjects with ELISA plasma Exosomes were measured by the sandwich ELISA method of anti-CD9 antibody-anti-CD9 antibody by the same method as in Comparative Example 1, and a significant difference test between Parkinson's disease patients and healthy subjects was performed. The p value was calculated. The obtained results are shown in Table 5 below.
  • Example 11 Evaluation of Parkinson's disease sample using the ratio of the amount of PS and the amount of exosomes having CD9 to the amount of exosomes having CD9 "Sample measurement value" obtained in Example 10 (Tim protein-anti-CD9 antibody sandwich ELISA method). By dividing (value (A)) by the “sample measurement value” (value (B)) obtained in Comparative Example 7 (anti-CD9 antibody-anti-CD9 antibody sandwich ELISA method), (A) / ( B) (hereinafter referred to as "corrected sample measurement value”) was obtained. Based on the obtained adjusted sample measurement values, a significant difference test between Parkinson's disease patients and healthy subjects was performed by the same method as in Example 1 (4), and AUC and p values were calculated.
  • FIG. 5 a box-and-whisker graph created based on the sample measurement values is shown in FIG.
  • the vertical axis shows the adjusted sample measurement value
  • the horizontal axis shows the result of a healthy person
  • PD shows the result of a Parkinson's disease patient.
  • the samples of Parkinson's disease patients with cognitive symptoms (MMSE of 27 or less) and plasmas derived from healthy subjects are used as samples, and the sample measurement values (values (A)) of exosomes having phosphatidylserine and CD9 have CD9.
  • the AUC is 0.804, which is a high accuracy. It was found that Parkinson's disease (particularly Parkinson's disease with cognitive symptoms) can be detected.
  • the sample measurements (value (A)) of exosomes having phosphatidylserine and CD9 were taken from Parkinson's disease patients without cognitive symptoms (MMSE greater than 27) and plasma from healthy subjects, respectively, and CD9 was used.
  • the corrected sample measurement value ((A) / (B)) obtained by dividing by the sample measurement value (value (B)) of the exosome possessed is evaluated, the AUC is 0.950, which is extremely high accuracy. It was found that Parkinson's disease (particularly Parkinson's disease without cognitive symptoms) can be detected.
  • the correlation coefficient between the corrected sample measurement value ((A) / (B)) and the MMSE was 0.7833, and a high correlation was observed.
  • Parkinson's disease is evaluated using plasma derived from Parkinson's disease patients (including Parkinson's disease patients with cognitive symptoms and Parkinson's disease patients without cognitive symptoms) and healthy subjects as samples, and the amount of exosomes having CD9 as an index.
  • AUC was obtained by dividing the sample measurement value (value (A)) of an exosome having 0.764, phosphatidylserine and CD9 by the sample measurement value (value (B)) of an exosome having CD9.
  • the corrected sample measurement values ((A) / (B)) were evaluated, the AUC was 0.877, and it was found that Parkinson's disease could be detected with high accuracy.

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Abstract

La présente invention aborde le problème consistant à fournir : un procédé destiné à aider au diagnostic de la maladie de Parkinson d'une manière simple ; un biomarqueur ; un kit de réactifs ; et un dispositif.　La présente invention concerne : un procédé d'aide au diagnostic de la maladie de Parkinson, le procédé comprenant les étapes consistant à mesurer la teneur en vésicule extracellulaire comportant de la phosphatidylsérine et de la tétraspanine ou à la fois la teneur en vésicule extracellulaire comportant de la phosphatidylsérine et de la tétraspanine et la teneur en vésicule extracellulaire comportant de la tétraspanine, et déterminer qu'un sujet souffre de la maladie de Parkinson en utilisant, en tant que critère, la teneur en vésicule extracellulaire comportant de la phosphatidylsérine et de la tétraspanine ou le rapport entre la teneur en vésicule extracellulaire comportant de la phosphatidylsérine et de la tétraspanine et la teneur en vésicule extracellulaire comportant de la tétraspanine ; un biomarqueur ; un kit de réactifs ; et un dispositif.
PCT/JP2020/044400 2019-11-29 2020-11-27 Procédé d'aide au diagnostic de la maladie de parkinson, biomarqueur, kit de réactifs et dispositif WO2021107155A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022250009A1 (fr) * 2021-05-26 2022-12-01 富士フイルム株式会社 Méthode d'aide à la différenciation de la maladie à corps de lewy et de la maladie d'alzheimer, biomarqueur et kit de réactifs

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016088689A1 (fr) 2014-12-05 2016-06-09 和光純薬工業株式会社 Transporteur lié à la protéine tim, procédés d'obtention, d'élimination et de détection de vésicules membranaires extracellulaires et de virus à l'aide dudit transporteur et kit comprenant ledit transporteur
JP2016161480A (ja) * 2015-03-04 2016-09-05 国立大学法人 千葉大学 微量の微小胞の定量、診断及びスクリーニング方法
WO2017032871A1 (fr) * 2015-08-27 2017-03-02 Georg-August-Universitaet Goettingen Stiftung Oeffentlichen Rechts, Universitaetsmedizin Procédé de diagnostic différentiel de la démence à corps de lewy et de la maladie de parkinson
JP2017520760A (ja) 2014-06-03 2017-07-27 レイセオン カンパニー アナログｒｆメモリシステム
JP2017525976A (ja) * 2014-06-27 2017-09-07 エックスワイ エバーグリーン テクノロジー カンパニー Ｃｎｓ由来エクソソームを富化するための方法
WO2018145211A1 (fr) * 2017-02-10 2018-08-16 UNIVERSITé LAVAL Vésicules extracellulaires dérivées d'érythrocytes et protéines associées à de telles vésicules en tant que biomarqueurs de la maladie de parkinson

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9005888B2 (en) * 2012-06-14 2015-04-14 System Biosciences, Llc Methods for microvesicle isolation and selective removal
US10914748B2 (en) * 2016-09-08 2021-02-09 UNIVERSITé LAVAL Erythrocyte-derived extracellular vesicles as a biomarker for clinically assessing Parkinson's disease
CA2941460C (fr) * 2016-09-08 2023-05-23 Universite Laval Vesicules extracellulaires derivees d'erythrocyte comme biomarqueur d'evaluation clinique de maladie de parkinson
US20180080945A1 (en) * 2016-09-20 2018-03-22 Nanosomix, Inc. Biomarkers and diagnostic methods for alzheimer's disease and other neurodegenerative disorders
JP7399712B2 (ja) * 2017-05-24 2023-12-18 ナノソミックス・インコーポレイテッド 疾病および障害の診断および予後判定のための小胞上のバイオマーカーの検出
CN115176163A (zh) * 2019-11-29 2022-10-11 富士胶片和光纯药株式会社 辅助阿尔茨海默型痴呆症或轻度认知障碍的诊断的方法、生物标志物、试剂盒及装置

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017520760A (ja) 2014-06-03 2017-07-27 レイセオン カンパニー アナログｒｆメモリシステム
JP2017525976A (ja) * 2014-06-27 2017-09-07 エックスワイ エバーグリーン テクノロジー カンパニー Ｃｎｓ由来エクソソームを富化するための方法
WO2016088689A1 (fr) 2014-12-05 2016-06-09 和光純薬工業株式会社 Transporteur lié à la protéine tim, procédés d'obtention, d'élimination et de détection de vésicules membranaires extracellulaires et de virus à l'aide dudit transporteur et kit comprenant ledit transporteur
JP2016161480A (ja) * 2015-03-04 2016-09-05 国立大学法人 千葉大学 微量の微小胞の定量、診断及びスクリーニング方法
WO2017032871A1 (fr) * 2015-08-27 2017-03-02 Georg-August-Universitaet Goettingen Stiftung Oeffentlichen Rechts, Universitaetsmedizin Procédé de diagnostic différentiel de la démence à corps de lewy et de la maladie de parkinson
WO2018145211A1 (fr) * 2017-02-10 2018-08-16 UNIVERSITé LAVAL Vésicules extracellulaires dérivées d'érythrocytes et protéines associées à de telles vésicules en tant que biomarqueurs de la maladie de parkinson

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Immunoassay", 2014, KODANSHA LTD.
MUSTAPIC M ET AL., FRONT. NEUROSCI., vol. 11, 2017, pages 278
NAOTO AOKI ET AL., STUDY OF OBESITY, vol. 13, no. 2, 2007
NATURE REVIEWS IMMUNOLOGY, vol. 9, August 2009 (2009-08-01), pages 581 - 593
OHMICHI, TAKUMA ET AL.: "Quantification of brain- derived extracellular vesicles in plasma as a biomarker to diagnose Parkinson's diseases", PARKINSONISM AND RELATED DISORDERS, vol. 61, April 2019 (2019-04-01), pages 82 - 87, XP085669225, DOI: 10.1016/j.parkreldis.2018.11.021 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022250009A1 (fr) * 2021-05-26 2022-12-01 富士フイルム株式会社 Méthode d'aide à la différenciation de la maladie à corps de lewy et de la maladie d'alzheimer, biomarqueur et kit de réactifs

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JP7348604B2 (ja) 2023-09-21
US20220283146A1 (en) 2022-09-08
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JPWO2021107155A1 (fr) 2021-06-03
EP4067905A1 (fr) 2022-10-05

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